Novel compositions and methods for the identification, assessment, prevention, and therapy of human cancers

ABSTRACT

The present invention is directed to the identification of markers that can be used to determine whether tumors are sensitive or resistant to a therapeutic agent. The present invention is also directed to the identification of therapeutic targets. The invention features a number of “sensitivity markers.” These are markers that are expressed in most or all cell lines that are sensitive to treatment with an agent and which are not expressed (or are expressed at a rather low level) in cells that are resistant to treatment with that agent. The invention also features a number of “resistance markers.” These are markers that are expressed in most or all cell lines that are resistant to treatment with an agent and which are not expressed (or are expressed at a rather low level) in cells that are sensitive to treatment with that agent.

RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 10/125,159, filed on Apr. 18, 2002, which claimsthe benefit of U.S. provisional patent application Ser. No. 60/295,031filed on May 31, 2001 and from U.S. provisional patent application Ser.No. 60/284, 773, filed on Apr. 18, 2001, and U.S. provisional patentapplication Ser. No. 60/ 284,764, filed on Apr. 18, 2001, which wasabandoned on May 30, 2001. The entire contents of each of theabove-identified applications are expressly incorporated herein byreference.

BACKGROUND OF THE INVENTION

Cancers can be viewed as a breakdown in the communication between tumorcells and their environment, including their normal neighboring cells.Growth-stimulatory and growth-inhibitory signals are routinely exchangedbetween cells within a tissue. Normally, cells do not divide in theabsence of stimulatory signals or in the presence of inhibitory signals.In a cancerous or neoplastic state, a cell acquires the ability to“override” these signals and to proliferate under conditions in which anormal cell would not.

In general, tumor cells must acquire a number of distinct aberranttraits in order to proliferate in an abnormal manner. Reflecting thisrequirement is the fact that the genomes of certain well-studied tumorscarry several different independently altered genes, including activatedoncogenes and inactivated tumor suppressor genes. In addition toabnormal cell proliferation, cells must acquire several other traits fortumor progression to occur. For example, early on in tumor progression,cells must evade the host immune system. Further, as tumor massincreases, the tumor must acquire vasculature to supply nourishment andremove metabolic waste. Additionally, cells must acquire an ability toinvade adjacent tissue. In many cases cells ultimately acquire thecapacity to metastasize to distant sites.

It is apparent that the complex process of tumor development and growthmust involve multiple gene products. It is therefore important to definethe role of specific genes involved in tumor development and growth andidentify those genes and gene products that can serve as targets for thediagnosis, prevention and treatment of cancers.

In the realm of cancer therapy it often happens that a therapeutic agentthat is initially effective for a given patient becomes, over time,ineffective or less effective for that patient. The very sametherapeutic agent may continue to be effective over a long period oftime for a different patient. Further, a therapeutic agent that iseffective, at least initially, for some patients can be completelyineffective or even harmful for other patients. Accordingly, it would beuseful to identify genes and/or gene products that represent prognosticgenes with respect to a given therapeutic agent or class of therapeuticagents. It then may be possible to determine which patients will benefitfrom particular therapeutic regimen and, importantly, determine when, ifever, the therapeutic regime begins to lose its effectiveness for agiven patient. The ability to make such predictions would make itpossible to discontinue a therapeutic regime that has lost itseffectiveness well before its loss of effectiveness becomes apparent byconventional measures.

SUMMARY OF THE INVENTION

The present invention is directed to the identification of markers thatcan be used to determine the sensitivity or resistance of tumors to atherapeutic agent. By examining the expression of one or more of theidentified markers, whose expression correlates with sensitivity to atherapeutic agent or resistance to a therapeutic agent, in a sample oftumor cells, it is possible to determine whether a therapeutic agent orcombination of agents will be most likely to reduce the growth rate ofthe tumor cells and can further be used in selecting appropriatetreatment agents. The markers of the present invention whose expressioncorrelates with sensitivity to an agent are listed in Tables 1 and 3(SEQ ID NOS:1-53 and Markers 59-124, respectively) and those whoseexpression correlates with resistance to an agent are listed in Tables 2and 4 (SEQ ID NOS:54-88 and Markers 1-58, respectively).

By examining the expression of one or more of the identified markers ina tumor, it is possible to determine which therapeutic agent orcombination of agents will be most likely to reduce the growth rate ofthe tumor. By examining the expression of one or more of the identifiedmarkers in a tumor, it is also possible to determine which therapeuticagent or combination of agents will be the least likely to reduce thegrowth rate of the tumor. By examining the expression of one or more ofthe identified markers, it is therefore possible to eliminateineffective or inappropriate therapeutic agents. Moreover, by examiningthe expression of one or more of the identified markers in a tumorsample taken from a patient during the course of therapeutic treatment,it is possible to determine whether the therapeutic treatment iscontinuing to be effective or whether the tumor has become resistant(refractory) to the therapeutic treatment. It is also possible toidentify new anti-cancer agents by examining the expression of one ormore markers when tumor cells are exposed to a potential anti-canceragent. Importantly, these determinations can be made on a patient bypatient basis or on an agent by agent (or combination of agents) basis.Thus, one can determine whether or not a particular therapeutictreatment is likely to benefit a particular patient or group/class ofpatients, or whether a particular treatment should be continued.

The present invention further provides previously unknown orunrecognized targets for the development of anti-cancer agents, such aschemotherapeutic compounds. The markers of the present invention can beused as targets in developing treatments (either single agent ormultiple agent) for cancer, particularly for those cancers which displayresistance to agents and exhibit expression of one or more of themarkers identified herein.

Other features and advantages of the invention will be apparent from thedetailed description and from the claims. Although materials and methodssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, the preferred materials andmethods are described below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the identification ofmarkers that can be used to determine whether a tumor is sensitive orresistant to a therapeutic agent. Based on these identifications, thepresent invention provides, without limitation: 1) methods fordetermining whether a therapeutic agent (or combination of agents) willor will not be effective in stopping or slowing tumor growth; 2) methodsfor monitoring the effectiveness of a therapeutic agent (or combinationof agents) used for the treatment of cancer; 3) methods for identifyingnew therapeutic agents for the treatment of cancer; 4) methods foridentifying combinations of therapeutic agents for use in treatingcancer; and 5) methods for identifying specific therapeutic agents andcombinations of therapeutic agents that are effective for the treatmentof cancer in specific patients.

Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patent applications,patents, database records, and other references mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions, will control. Inaddition, the materials, methods, and examples are illustrative only andare not intended to be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

A “marker” is a naturally-occurring polymer corresponding to at leastone of the nucleic acids listed in Tables 1-4. For example, markersinclude, without limitation, sense and anti-sense strands of genomic DNA(i.e. including any introns occurring therein), RNA generated bytranscription of genomic DNA (i.e. prior to splicing), RNA generated bysplicing of RNA transcribed from genomic DNA, and proteins generated bytranslation of spliced RNA (i.e. including proteins both before andafter cleavage of normally cleaved regions such as transmembrane signalsequences). As used herein, “marker” may also include a cDNA made byreverse transcription of an RNA generated by transcription of genomicDNA (including spliced RNA).

The term “probe” refers to any molecule which is capable of selectivelybinding to a specifically intended target molecule, for example a markerof the invention. Probes can be either synthesized by one skilled in theart, or derived from appropriate biological preparations. For purposesof detection of the target molecule, probes may be specifically designedto be labeled, as described herein. Examples of molecules that can beutilized as probes include, but are not limited to, RNA, DNA, proteins,antibodies, and organic monomers.

The “normal” level of expression of a marker is the level of expressionof the marker in cells of a patient not afflicted with cancer.

“Over-expression” and “under-expression” of a marker refer to expressionof the marker of a patient at a greater or lesser level, respectively,than normal level of expression of the marker (e.g. at least two-foldgreater or lesser level).

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue-specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cell under mostor all physiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

A “transcribed polynucleotide” is a polynucleotide (e.g. an RNA, a cDNA,or an analog of one of an RNA or cDNA) which is complementary to orhomologous with all or a portion of a mature RNA made by transcriptionof a genomic DNA corresponding to a marker of the invention and normalpost-transcriptional processing (e.g. splicing), if any, of thetranscript.

“Complementary” refers to the broad concept of sequence complementaritybetween regions of two nucleic acid strands or between two regions ofthe same nucleic acid strand. It is known that an adenine residue of afirst nucleic acid region is capable of forming specific hydrogen bonds(“base pairing”) with a residue of a second nucleic acid region which isantiparallel to the first region if the residue is thymine or uracil.Similarly, it is known that a cytosine residue of a first nucleic acidstrand is capable of base pairing with a residue of a second nucleicacid strand which is antiparallel to the first strand if the residue isguanine. A first region of a nucleic acid is complementary to a secondregion of the same or a different nucleic acid if, when the two regionsare arranged in an antiparallel fashion, at least one nucleotide residueof the first region is capable of base pairing with a residue of thesecond region. Preferably, the first region comprises a first portionand the second region comprises a second portion, whereby, when thefirst and second portions are arranged in an antiparallel fashion, atleast about 50%, and preferably at least about 75%, at least about 90%,or at least about 95% of the nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion. More preferably, all nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion.

“Homologous” as used herein, refers to nucleotide sequence similaritybetween two regions of the same nucleic acid strand or between regionsof two different nucleic acid strands. When a nucleotide residueposition in both regions is occupied by the same nucleotide residue,then the regions are homologous at that position. A first region ishomologous to a second region if at least one nucleotide residueposition of each region is occupied by the same residue. Homologybetween two regions is expressed in terms of the proportion ofnucleotide residue positions of the two regions that are occupied by thesame nucleotide residue. By way of example, a region having thenucleotide sequence 5′-ATTGCC-3′ and a region having the nucleotidesequence 5′-TATGGC-3′ share 50% homology. Preferably, the first regioncomprises a first portion and the second region comprises a secondportion, whereby, at least about 50%, and preferably at least about 75%,at least about 90%, or at least about 95% of the nucleotide residuepositions of each of the portions are occupied by the same nucleotideresidue. More preferably, all nucleotide residue positions of each ofthe portions are occupied by the same nucleotide residue.

A marker is “fixed” to a substrate if it is covalently or non-covalentlyassociated with the substrate such the substrate can be rinsed with afluid (e.g. standard saline citrate, pH 7.4) without a substantialfraction of the marker dissociating from the substrate.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g. encodes a natural protein).

Expression of a marker in a patient is “significantly” higher or lowerthan the normal level of expression of a marker if the level ofexpression of the marker is greater or less, respectively, than thenormal level by an amount greater than the standard error of the assayemployed to assess expression, and preferably at least twice, and morepreferably three, four, five or ten times that amount. Alternately,expression of the marker in the patient can be considered“significantly” higher or lower than the normal level of expression ifthe level of expression is at least about two, and preferably at leastabout three, four, or five times, higher or lower, respectively, thanthe normal level of expression of the marker.

Cancer is “inhibited” if at least one symptom of the cancer isalleviated, terminated, slowed, or prevented. As used herein, cancer isalso “inhibited” if recurrence or metastasis of the cancer is reduced,slowed, delayed, or prevented.

A tumor, including tumor cells, is “sensitive” (Tables 1 and 3) to atherapeutic agent if its rate of growth is inhibited as a result ofcontact with the therapeutic agent, compared to its growth in theabsence of contact with the therapeutic agent. The quality of beingsensitive to a therapeutic agent is a variable one, with differenttumors exhibiting different levels of “sensitivity” to a giventherapeutic agent, under different conditions. In one embodiment of theinvention, tumors may be predisposed to sensitivity to an agent if oneor more of the corresponding sensitivity markers are expressed.

A tumor, including tumor cells, is “resistant” (Tables 2 and 4) to atherapeutic agent if its rate of growth is not inhibited, or inhibitedto a very low degree, as a result of contact with the therapeutic agentwhen compared to its growth in the absence of contact with thetherapeutic agent. The quality of being resistant to a therapeutic agentis a highly variable one, with different tumors exhibiting differentlevels of“resistance” to a given therapeutic agent, under differentconditions. In another embodiments of the invention, tumors may bepredisposed to resistance to an agent if one or more of thecorresponding resistance markers are expressed.

A kit is any manufacture (e.g. a package or container) comprising atleast one reagent, e.g. a probe, for specifically detecting a marker ofthe invention. The manufacture may be promoted, distributed, or sold asa unit for performing the methods of the present invention. The reagentsincluded in such a kit comprise probes/primers and/or antibodies for usein detecting sensitivity and resistance gene expression. In addition,the kits of the present invention may preferably contain instructionswhich describe a suitable detection assay. Such kits can be convenientlyused, e.g., in clinical settings, to diagnose patients exhibitingsymptoms of cancer, in particular patients exhibiting the possiblepresence of a tumor.

Specific Embodiments

1. Identification Of Sensitivity And Resistance Genes

The present invention provides genes that are expressed in tumors thatare sensitive to a given therapeutic agent and whose expressioncorrelates with sensitivity to that therapeutic agent (Tables 1 and 3).The present invention also provides genes that are expressed in tumorsthat are resistant to a given therapeutic agent and whose expressioncorrelates with resistance to that therapeutic agent (Tables 2 and 4).The present invention further provides novel genes (Tables 1 and 2, SEQID NOS: 1-88). Accordingly, one or more of the genes of the presentinvention can be used as markers (or surrogate markers) to identifytumors, including tumor cells, that are likely to be successfullytreated by that agent. In addition, the markers of the present inventioncan be used to identify cancers that have become or are at risk ofbecoming refractory to treatment with the agent.

Tables 1 and 3 show markers whose expression correlates with sensitivityto a taxane compound and/or a platinum compound (SEQ ID NOS: 1-53 andMarkers 59-124, respectively). Tables 2 and 4 show markers whoseexpression correlates with resistance to a taxane compound and/or aplatinum compound (SEQ ID NOS:54-88 and Markers 1-58, respectively). Inparticular, the Tables set forth markers identified in ovarian tumorsamples as sensitive or resistant to therapy with TAXOL and cisplatin.

II. Determining Sensitivity or Resistance To An Agent

The expression level of the identified sensitivity and resistance genes,or the proteins encoded by the identified sensitivity and resistancegenes, may be used to: 1) determine if a tumor can be or is likely to besuccessfully treated by an agent or combination of agents; 2) determineif a tumor is responding to treatment with an agent or combination ofagents; 3) select an appropriate agent or combination of agents fortreating a tumor; 4) monitor the effectiveness of an ongoing treatment;and 5) identify new treatments (either single agent or combination ofagents). In particular, the identified sensitivity and resistance genesmay be utilized as markers (surrogate and/or direct) to determineappropriate therapy, to monitor clinical therapy and human trials of adrug being tested for efficacy, and to develop new agents andtherapeutic combinations.

Accordingly, the present invention provides methods for determiningwhether an agent, e.g., a chemotherapeutic agent, can be used to reducethe growth rate of a tumor comprising the steps of:

-   -   a) obtaining a sample of tumor cells;    -   b) determining whether the tumor cells express one or more        markers identified in Tables 1-4; and    -   c) identifying that an agent is or is not appropriate to treat        the tumor based on the expression of one or more markers        identified in Tables 1-4.

In another embodiment, the invention provides a method for determiningwhether an agent can be used to reduce the growth of a tumor, comprisingthe steps of:

-   -   a) obtaining a sample of tumor cells;    -   b) determining whether the tumor cells express one or more        markers identified in Tables 1-4; and    -   c) identifying that an agent is appropriate to treat the tumor        when the expression of the sensitivity markers identified in        Tables 1 and 3 is increased and/or the expression of the        resistance markers identified in Tables 2 and 4 is decreased in        the presence of the agent.

Alternatively, in step (c), an agent can be identified as not beingappropriate to treat the tumor when the expression of the sensitivitymarkers identified in Tables 1 and 3 is decreased and/or the expressionof the resistance markers identified in Tables 2 and 4 is increased inthe presence of the agent.

In another embodiment, the invention provides a method for determiningwhether an agent can be used to reduce the growth of a tumor, comprisingthe steps of:

-   -   a) obtaining a sample of tumor cells;    -   b) exposing some of the tumor cells to one or more test agents;    -   c) determining the level of expression of one or more markers        identified in Tables 1-4 both in tumor cells exposed to the        agent and in tumor cells that have not been exposed to the        agent; and    -   d) identifying that an agent is appropriate to treat the tumor        when the expression of the sensitivity markers identified in        Tables 1 and 3 is increased and/or the expression of the        resistance markers identified in Tables 2 and 4 is decreased in        the presence of the agent.

Alternatively, in step (d), an agent can be identified as not beingappropriate to treat the tumor when the expression of the sensitivitymarkers identified in Tables 1 and 3 is decreased and/or the expressionof the resistance markers identified in Tables 2 and 4 is increased inthe presence of the agent.

In another embodiment, the invention provides a method for determiningwhether treatment with an anti-cancer agent should be continued in acancer patient, comprising the steps of:

-   -   a) obtaining two or more samples of tumors cells from a patient        at different times during the course of anti-cancer agent        treatment;    -   b) determining the level of expression in the tumors cells of        one or more genes which correspond to markers identified in        Tables 1-4 in the two or more samples; and    -   c) continuing the treatment when the expression level of the        sensitivity markers identified in Tables I and 3 does not        decrease and/or the expression level of the resistance markers        identified in Tables 2 and 4 does not increase during the course        of treatment.

Alternatively, in step (c), the treatment is discontinued when theexpression level of the sensitivity markers identified in Tables 1 and 3is decreased and/or the expression level of the resistance markersidentified in Tables 2 and 4 is increased, during the course oftreatment.

The markers of the present invention are predictive of chemotherapeuticagents, generally. In one embodiment of the invention, the agents usedin methods of the invention is a taxane compound. In another embodiment,the agent is a platinum compound. In yet another embodiment, the agentis a combination of a taxane compound and a platinum compound e.g.,TAXOL and cisplatin, respectively.

In another embodiment of the invention, the expression of genes whichcorrespond to markers identified in Tables 1-4 is detected by measuringmRNA which corresponds to the gene. In yet another embodiment of theinvention, the expression of genes which correspond to markersidentified in Tables 1-4 is detected by measuring protein whichcorresponds to the gene. In a further another embodiment of theinvention, the tumor cells used in the methods of the invention areobtained from a patient.

In another embodiment, the invention provides a method of treating apatient for cancer by administering to the patient an agent which hasbeen identified as being effective against cancer by methods of theinvention described herein.

As used herein, the term “agent” is defined broadly as anything thatcancer cells, including tumor cells, may be exposed to in a therapeuticprotocol. In the context of the present invention, such agents include,but are not limited to, chemotherapeutic agents, such as anti-metabolicagents, e.g., Ara AC, 5-FU and methotrexate, antimitotic agents, e.g.,TAXOL, inblastine and vincristine, alkylating agents, e.g., melphanlan,BCNU and nitrogen mustard, Topoisomerase II inhibitors, e.g., VW-26,topotecan and Bleomycin, strand-breaking agents, e.g., doxorubicin andDHAD, cross-linking agents, e.g., cisplatin and CBDCA, radiation andultraviolet light. In a preferred embodiment, the agent is a taxanecompound (e.g., TAXOL) and/or a platinum compound (e.g., cisplatin).

Further to the above, the language “chemotherapeutic agent” is intendedto include chemical reagents which inhibit the growth of proliferatingcells or tissues wherein the growth of such cells or tissues isundesirable. Chemotherapeutic agents are well known in the art (seee.g., Gilman A. G., et al., The Pharmacological Basis of Therapeutics,8th Ed., Sec 12:1202-1263 (1990)), and are typically used to treatneoplastic diseases. The chemotherapeutic agents generally employed inchemotherapy treatments are listed below in Table A. TABLE ANONPROPRIETARY NAMES CLASS TYPE OF AGENT (OTHER NAMES) AlkylatingNitrogen Mustards Mechlorethamine (HN₂) Cyclophosphamide IfosfamideMelphalan (L-sarcolysin) Chlorambucil Ethylenimines HexamethylmelamineAnd Methylmelamines Thiotepa Alkyl Sulfonates Busulfan AlkylatingNitrosoureas Carmustine (BCNU) Lomustine (CCNU) Semustine (methyl-CCNU)Streptozocin (streptozotocin) Triazenes Decarbazine (DTIC;dimethyltriazeno- imidazolecarboxamide) Alkylator cis-diamminedichloro-platinum II (CDDP) Antimetabolites Folic Acid Methotrexate Analogs(amethopterin) Pyrimidine Fluorouracil Analogs (′5-fluorouracil; 5-FU)Floxuridine (fluorode- oxyuridine; FUdR) Cytarabine (cytosinearabinoside) Purine Analogs Mercaptopuine and Related (6-mercaptopurine;Inhibitors 6-MP) Thioguanine (6-thioguanine; TG) Pentostatin(2′-deoxycoformycin) Vinca Alkaloids Vinblastin (VLB) VincristineTopoisomerase Etoposide Inhibitors Teniposide Camptothecin Topotecan9-amino-campotothecin CPT-11 Natural Antibiotics Dactinomycin Products(actinomycin D) Adriamycin Daunorubicin (daunomycin; rubindomycin)Doxorubicin Bleomycin Plicamycin (mithramycin) Mitomycin (mitomycin C)TAXOL Taxotere Enzymes L-Asparaginase Biological Interfon alfa Responseinterleukin 2 Modifiers Miscellaneous Platinum cis-diamminedichloro-Agents Coordination platinum II (CDDP) Complexes CarboplatinAnthracendione Mitoxantrone Substituted Urea Hydroxyurea MethylHydraxzine Procarbazine Derivative (N-methylhydrazine, (MIH)Adrenocortical Mitotane (o,p′-DDD) Suppressant AminoglutethimideHormones and Adrenocorticosteroids Prednisone Antagonists ProgestinsHydroxyprogesterone caproate Medroxyprogesterone acetate Megestrolacetate Estrogens Diethylstilbestrol Ethinyl estradiol AntiestrogenTamoxifen Androgens Testosterone propionate Fluoxymesterone AntiandrogenFlutamide Gonadotropin-releasing Leuprolide Hormone analog

The agents tested in the present methods can be a single agent or acombination of agents. For example, the present methods can be used todetermine whether a single chemotherapeutic agent, such as methotrexate,can be used to treat a cancer or whether a combination of two or moreagents can be used. Preferred combinations will include agents that havedifferent mechanisms of action, e.g., the use of an anti-mitotic agentin combination with an alkylating agent.

As used herein, cancer cells, including tumor cells, refer to cells thatdivide at an abnormal (increased) rate. Cancer cells include, but arenot limited to, carcinomas, such as squamous cell carcinoma, basal cellcarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,adenocarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma,bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-livercell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillarycarcinoma, transitional cell carcinoma, choriocarcinoma, semonoma,embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma,colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamouscell carcinoma of the neck and head region; sarcomas, such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordosarcoma, angiosarcoma, endotlieliosarcoma,lymphangiosarcoma, synoviosarcoma and mesotheliosarcoma; leukemias andlymphomas such as granulocytic leukemia, monocytic leukemia, lymphocyticleukemia, malignant lymphoma, plasmocytoma, reticulum cell sarcoma, orHodgkins disease; and tumors of the nervous system including glioma,meningoma, medulloblastoma, schwannoma or epidymoma.

The source of the cancer cells used in the present method will be basedon how the method of the present invention is being used. For example,if the method is being used to determine whether a patient's cancer canbe treated with an agent, or a combination of agents, then the preferredsource of cancer cells will be cancer cells obtained from a cancerbiopsy from the patient, e.g., a tumor biopsy. Alternatively, a cancercell line similar to the type of cancer being treated can be assayed.For example if ovarian cancer is being treated, then an ovarian cancercell line can be used. If the method is being used to monitor theeffectiveness of a therapeutic protocol, then a tissue sample from thepatient being treated is the preferred source. If the method is beingused to identify new therapeutic agents or combinations, any cancercells, e.g., cells of a cancer cell line, can be used.

A skilled artisan can readily select and obtain the appropriate cancercells that are used in the present method. For cancer cell lines,sources such as The National Cancer Institute, for the NCI-60 cells, arepreferred. For cancer cells obtained from a patient, standard biopsymethods, such as a needle biopsy, can be employed.

Ovarian tumor samples were used to obtain the markers of the presentinvention. It will thus be appreciated that cells from ovarian tumorsare particularly useful in the methods of the present invention.

In the methods of the present invention, the level or amount ofexpression of one or more genes selected from the group consisting ofthe genes identified in Tables 1-4 is determined. As used herein, thelevel or amount of expression refers to the absolute level of expressionof an mRNA encoded by the gene or the absolute level of expression ofthe protein encoded by the gene (i.e., whether or not expression is oris not occurring in the cancer cells).

Generally, it is preferable to determine the expression of two or moreof the identified sensitivity or resistance genes, more preferably,three or more of the identified sensitivity or resistance genes, mostpreferably, a set of the identified sensitivity and/or resistance genes.Thus, it is preferable to assess the expression of a panel ofsensitivity and resistance genes.

As an alternative to making determinations based on the absoluteexpression level of selected genes, determinations may be based on thenormalized expression levels. Expression levels are normalized bycorrecting the absolute expression level of a sensitivity or resistancegene by comparing its expression to the expression of a gene that is nota sensitivity or resistance gene, e.g., a housekeeping genes that isconstitutively expressed. Suitable genes for normalization includehousekeeping genes such as the actin gene. This normalization allows oneto compare the expression level in one sample, e.g., a tumor sample, toanother sample, e.g., a non-tumor sample, or between samples fromdifferent sources.

Alternatively, the expression level can be provided as a relativeexpression level. To determine a relative expression level of a gene,the level of expression of the gene is determined for 10 or moresamples, preferably 50 or more samples, prior to the determination ofthe expression level for the sample in question. The mean expressionlevel of each of the genes assayed in the larger number of samples isdetermined and this is used as a baseline expression level for thegene(s) in question. The expression level of the gene determined for thetest sample (absolute level of expression) is then divided by the meanexpression value obtained for that gene. This provides a relativeexpression level and aids in identifying extreme cases of sensitivity orresistance.

Preferably, the samples used will be from similar tumors or fromnon-cancerous cells of the same tissue origin as the tumor in question.The choice of the cell source is dependent on the use of the relativeexpression level data. For example, using tumors of similar types forobtaining a mean expression score allows for the identification ofextreme cases of sensitivity or resistance. Using expression found innormal tissues as a mean expression score aids in validating whether thesensitivity/resistance gene assayed is tumor specific (versus normalcells). Such a later use is particularly important in identifyingwhether a sensitivity or resistance gene can serve as a target gene. Inaddition, as more data is accumulated, the mean expression value can berevised, providing improved relative expression values based onaccumulated data.

III. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat correspond to a marker of the invention, in particular, the novelnucleic acid molecules set forth in Tables I and 2 as SEQ ID NOS:1-88.The isolated nucleic acid molecules of the present invention includenucleic acids which encode a polypeptide corresponding to a marker ofthe invention or a portion of such a polypeptide. Isolated nucleic acidsof the invention also include nucleic acid molecules sufficient for useas hybridization probes to identify nucleic acid molecules thatcorrespond to a marker of the invention, including nucleic acids whichencode a polypeptide corresponding to a marker of the invention, andfragments of such nucleic acid molecules, e.g., those suitable for useas PCR primers for the amplification or mutation of nucleic acidmolecules. As used herein, the term “nucleic acid molecule” is intendedto include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Preferably, an “isolated” nucleic acid moleculeis free of sequences (preferably protein-encoding sequences) whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kB, 4kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidencoding a protein corresponding to a marker listed in Tables 1-4, canbe isolated using standard molecular biology techniques and the sequenceinformation in the database records described herein. Using all or aportion of such nucleic acid sequences, nucleic acid molecules of theinvention can be isolated using standard hybridization and cloningtechniques (e.g., as described in Sambrook et al., ed., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989).

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA, or genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to all or a portion of a nucleic acid molecule of theinvention can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which has a nucleotidesequence complementary to the nucleotide sequence of a nucleic acidcorresponding to a marker of the invention or to the nucleotide sequenceof a nucleic acid encoding a protein which corresponds to a marker ofthe invention. A nucleic acid molecule which is complementary to a givennucleotide sequence is one which is sufficiently complementary to thegiven nucleotide sequence that it can hybridize to the given nucleotidesequence thereby forming a stable duplex.

Moreover, a nucleic acid molecule of the invention can comprise only aportion of a nucleic acid sequence, wherein the full length nucleic acidsequence comprises a marker of the invention or which encodes apolypeptide corresponding to a marker of the invention. Such nucleicacids can be used, for example, as a probe or primer. The probe/primertypically is used as one or more substantially purifiedoligonucleotides. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 7, preferably about 15, more preferably about 25, 50, 75,100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutivenucleotides of a nucleic acid of the invention.

Probes based on the sequence of a nucleic acid molecule of the inventioncan be used to detect transcripts or genomic sequences corresponding toone or more markers of the invention. The probe comprises a label groupattached thereto, e.g., a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used as part of adiagnostic test kit for identifying cells or tissues which mis-expressthe protein, such as by measuring levels of a nucleic acid moleculeencoding the protein in a sample of cells from a subject, e.g.,detecting mRNA levels or determining whether a gene encoding the proteinhas been mutated or deleted.

The invention further encompasses nucleic acid molecules that differ,due to degeneracy of the genetic code, from the nucleotide sequence ofnucleic acids encoding a protein which corresponds to a marker of theinvention, and thus encode the same protein.

In addition to the nucleotide sequences described in the databaserecords described herein, it will be appreciated by those skilled in theart that DNA sequence polymorphisms that lead to changes in the aminoacid sequence can exist within a population (e.g., the humanpopulation). Such genetic polymorphisms can exist among individualswithin a population due to natural allelic variation. An allele is oneof a group of genes which occur alternatively at a given genetic locus.In addition, it will be appreciated that DNA polymorphisms that affectRNA expression levels can also exist that may affect the overallexpression level of that gene (e.g., by affecting regulation ordegradation).

As used herein, the phrase “allelic variant” refers to a nucleotidesequence which occurs at a given locus or to a polypeptide encoded bythe nucleotide sequence.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a polypeptidecorresponding to a marker of the invention. Such natural allelicvariations can typically result in 1-5% variance in the nucleotidesequence of a given gene. Alternative alleles can be identified bysequencing the gene of interest in a number of different individuals.This can be readily carried out by using hybridization probes toidentify the same genetic locus in a variety of individuals. Any and allsuch nucleotide variations and resulting amino acid polymorphisms orvariations that are the result of natural allelic variation and that donot alter the functional activity are intended to be within the scope ofthe invention.

In another embodiment, an isolated nucleic acid molecule of theinvention is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250,300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600,1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, or morenucleotides in length and hybridizes under stringent conditions to anucleic acid corresponding to a marker of the invention or to a nucleicacid encoding a protein corresponding to a marker of the invention. Asused herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 75% (80%, 85%, preferably 95%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in sections 6.3.1-6.3.6 of Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989). A preferred, non-limiting example ofstring ent hybridization conditions for annealing two single-strandedDNA each of which is at least about 100 bases in length and/or forannealing a single-stranded DNA and a single-stranded RNA each of whichis at least about 100 bases in length, are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1 % SDS at 50-65° C. Further preferredhybridization conditions are taught in Lockhart, et al., NatureBiotechnology, Volume 14, August 1996:1675-1680; Breslauer, et al.,Proc. Natl. Acad. Sci. USA, Volume 83, June 1986: 3746-3750; Van Ness,et al., Nucleic Acids Research, Volume 19, No. 19, September 1991:5143-5151; McGraw, et al., BioTechniques, Volume 8, No. 6 1990: 674-678;and Milner, et al., Nature Biotechnology, Volume 15, June 1997: 537-541,all expressly incorporated by reference.

In addition to naturally-occurring allelic variants of a nucleic acidmolecule of the invention that can exist in the population, the skilledartisan will further appreciate that sequence changes can be introducedby mutation thereby leading to changes in the amino acid sequence of theencoded protein, without altering the biological activity of the proteinencoded thereby. For example, one can make nucleotide substitutionsleading to amino acid substitutions at “non-essential” amino acidresidues. A “non-essential” amino acid residue is a residue that can bealtered from the wild-type sequence without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. For example, amino acid residues that are notconserved or only semi-conserved among homologs of various species maybe non-essential for activity and thus would be likely targets foralteration. Alternatively, amino acid residues that are conserved amongthe homologs of various species (e.g., murine and human) may beessential for activity and thus would not be likely targets foralteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding a polypeptide of the invention that contain changesin amino acid residues that are not essential for activity. Suchpolypeptides differ in amino acid sequence from the naturally-occurringproteins which correspond to the markers of the invention, yet retainbiological activity. In one embodiment, such a protein has an amino acidsequence that is at least about 40% identical, 50%, 60%, 70%, 80%, 90%,95%, or 98% identical to the amino acid sequence of one of the proteinswhich correspond to the markers of the invention.

An isolated nucleic acid molecule encoding a variant protein can becreated by introducing one or more nucleotide substitutions, additionsor deletions into the nucleotide sequence of nucleic acids of theinvention, such that one or more amino acid residue substitutions,additions, or deletions are introduced into the encoded protein.Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined.

The present invention encompasses antisense nucleic acid molecules,i.e., molecules which are complementary to a sense nucleic acid of theinvention, e.g., complementary to the coding strand of a double-strandedcDNA molecule corresponding to a marker of the invention orcomplementary to an mRNA sequence corresponding to a marker of theinvention. Accordingly, an antisense nucleic acid of the invention canhydrogen bond to (i.e. anneal with) a sense nucleic acid of theinvention. The antisense nucleic acid can be complementary to an entirecoding strand, or to only a portion thereof, e.g., all or part of theprotein coding region (or open reading frame). An antisense nucleic acidmolecule can also be antisense to all or part of a non-coding region ofthe coding strand of a nucleotide sequence encoding a polypeptide of theinvention. The non-coding regions (“5′ and 3′ untranslated regions”) arethe 5′ and 3′ sequences which flank the coding region and are nottranslated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45, or 50 or more nucleotides in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6-adenine,7-methyl guanine, 5-methyl aminomethyluracil,5-methoxyaminomethyl-2-thilouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-inethyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been sub-cloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a polypeptidecorresponding to a selected marker of the invention to thereby inhibitexpression of the marker, e.g., by inhibiting transcription and/ortranslation. The hybridization can be by conventional nucleotidecomplementarity to form a stable duplex, or, for example, in the case ofan antisense nucleic acid molecule which binds to DNA duplexes, throughspecific interactions in the major groove of the double helix. Examplesof a route of administration of antisense nucleic acid molecules of theinvention includes direct injection at a tissue site or infusion of theantisense nucleic acid into an ovary-associated body fluid.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense molecules can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g., by linking the antisense nucleic acidmolecules to peptides or antibodies which bind to cell surface receptorsor antigens. The antisense nucleic acid molecules can also be deliveredto cells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual α-units, the strands run parallel to each other(Gaultier et al., 1987, Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

The invention also encompasses ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes asdescribed in Haselhoff and Gerlach, 1988, Nature 334:585-591) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. A ribozyme havingspecificity for a nucleic acid molecule encoding a polypeptidecorresponding to a marker of the invention can be designed based uponthe nucleotide sequence of a cDNA corresponding to the marker. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved (see Cech et al. U.S. Pat. No.4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Alternatively, anmRNA encoding a polypeptide of the invention can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules (see, e.g., Bartel and Szostak, 1993, Science 261:1411-1418).

The invention also encompasses nucleic acid molecules which form triplehelical structures. For example, expression of a polypeptide of theinvention can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the gene encoding thepolypeptide (e.g., the promoter and/or enhancer) to form triple helicalstructures that prevent transcription of the gene in target cells. Seegenerally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992)Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays14(12):807-15.

In various embodiments, the nucleic acid molecules of the invention canbe modified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal., 1996, Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein,the terms “peptide nucleic acids” or “PNAs” refer to nucleic acidmimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670-675.

PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup (1996), supra; or as probes or primers for DNA sequence andhybridization (Hyrup, 1996, supra; Perry-O'Keefe et al., 1996, Proc.Natl. Acad. Sci. USA 93:14670-675).

In another embodiment, PNAs can be modified, e.g., to enhance theirstability or cellular uptake, by attaching lipophilic or other helpergroups to PNA, by the formation of PNA-DNA chimeras, or by the use ofliposomes or other techniques of drug delivery known in the art. Forexample, PNA-DNA chimeras can be generated which can combine theadvantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNASE H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup, 1996, supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry and modified nucleosideanalogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite can be used as a link between the PNA and the 5′ end ofDNA (Mag et al., 1989, Nucleic Acids Res. 17:5973-88). PNA monomers arethen coupled in a step-wise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn et al., 1996, Nucleic AcidsRes. 24(17):3357-63). Alternatively, chimeric molecules can besynthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al.,1975, Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide can include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al., 1988, Bio/Techniques 6:958-976) or intercalating agents (see,e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide can be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The invention also includes molecular beacon nucleic acids having atleast one region which is complementary to a nucleic acid of theinvention, such that the molecular beacon is useful for quantitating thepresence of the nucleic acid of the invention in a sample. A “molecularbeacon” nucleic acid is a nucleic acid comprising a pair ofcomplementary regions and having a fluorophore and a fluorescentquencher associated therewith. The fluorophore and quencher areassociated with different portions of the nucleic acid in such anorientation that when the complementary regions are annealed with oneanother, fluorescence of the fluorophore is quenched by the quencher.When the complementary regions of the nucleic acid are not annealed withone another, fluorescence of the fluorophore is quenched to a lesserdegree. Molecular beacon nucleic acids are described, for example, inU.S. Pat. No. 5,876,930.

IV. Isolated Proteins and Antibodies

One aspect of the invention pertains to isolated proteins whichcorrespond to individual markers of the invention, and biologicallyactive portions thereof, as well as polypeptide fragments suitable foruse as immunogens to raise antibodies directed against a polypeptidecorresponding to a marker of the invention. In one embodiment, thenative polypeptide corresponding to a marker can be isolated from cellsor tissue sources by an appropriate purification scheme using standardprotein purification techniques. In another embodiment, polypeptidescorresponding to a marker of the invention are produced by recombinantDNA techniques. Alternative to recombinant expression, a polypeptidecorresponding to a marker of the invention can be synthesized chemicallyusing standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”). When the protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When the protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals,.i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the proteinhave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest.

Biologically active portions of a polypeptide corresponding to a markerof the invention include polypeptides comprising amino acid sequencessufficiently identical to or derived from the amino acid sequence of theprotein corresponding to the marker, which include fewer amino acidsthan the full length protein, and exhibit at least one activity of thecorresponding full-length protein. Typically, biologically activeportions comprise a domain or motif with at least one activity of thecorresponding protein. A biologically active portion of a protein of theinvention can be a polypeptide which is, for example, 10, 25, 50, 100 ormore amino acids in length. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of the native form of a polypeptide of theinvention.

Preferred polypeptides have the amino acid sequence listed in the one ofthe GenBank and NUC database records described herein. Other usefulproteins are substantially identical (e.g., at least about 40%,preferably 50%, 60%, 70%, 80%, 90%, 95%, or 99%) to one of thesesequences and retain the functional activity of the protein of thecorresponding naturally-occurring protein yet differ in amino acidsequence due to natural allelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0) which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Yet another useful algorithm for identifying regions of local sequencesimilarity and alignment is the FASTA algorithm as described in Pearsonand Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. When usingthe FASTA algorithm for comparing nucleotide or amino acid sequences, aPAM120 weight residue table can, for example, be used with a k-tuplevalue of 2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

The invention also provides chimeric or fusion proteins corresponding toa marker of the invention. As used herein, a “chimeric protein” or“fusion protein” comprises all or part (preferably a biologically activepart) of a polypeptide corresponding to a marker of the inventionoperably linked to a heterologous polypeptide (i.e., a polypeptide otherthan the polypeptide corresponding to the marker). Within the fusionprotein, the term “operably linked” is intended to indicate that thepolypeptide of the invention and the heterologous polypeptide are fusedin-frame to each other.-The heterologous polypeptide can be fused to theamino-terminus or the carboxyl-terminus of the polypeptide of theinvention.

One useful fusion protein is a GST fusion protein in which a polypeptidecorresponding to a marker of the invention is fused to the carboxylterminus of GST sequences. Such fusion proteins can facilitate thepurification of a recombinant polypeptide of the invention.

In another embodiment, the fusion protein contains a heterologous signalsequence at its amino terminus. For example, the native signal sequenceof a polypeptide corresponding to a marker of the invention can beremoved and replaced with a signal sequence from another protein. Forexample, the gp67 secretory sequence of the baculovirus envelope proteincan be used as a heterologous signal sequence (Ausubel et al., ed.,Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1992).Other examples of eukaryotic heterologous signal sequences include thesecretory sequences of melittin and human placental alkaline phosphatase(Stratagene; La Jolla, Calif.). In yet another example, usefulprokaryotic heterologous signal sequences include the phoA secretorysignal (Sambrook et al., supra) and the protein A secretory signal(Pharmacia Biotech; Piscataway, N.J.).

In yet another embodiment, the fusion protein is an immunoglobulinfusion protein in which all or part of a polypeptide corresponding to amarker of the invention is fused to sequences derived from a member ofthe immunoglobulin protein family. The immunoglobulin fusion proteins ofthe invention can be incorporated into pharmaceutical compositions andadministered to a subject to inhibit an interaction between a ligand(soluble or membrane-bound) and a protein on the surface of a cell(receptor), to thereby suppress signal transduction in vivo. Theimmunoglobulin fusion protein can be used to affect the bioavailabilityof a cognate ligand of a polypeptide of the invention. Inhibition ofligand/receptor interaction can be useful therapeutically, both fortreating proliferative and differentiative disorders and for modulating(e.g. promoting or inhibiting) cell survival. Moreover, theimmunoglobulin fusion proteins of the invention can be used asimmunogens to produce antibodies directed against a polypeptide of theinvention in a subject, to purify ligands and in screening assays toidentify molecules which inhibit the interaction of receptors withligands.

Chimeric and fusion proteins of the invention can be produced bystandard recombinant DNA techniques. In another embodiment, the fusiongene can be synthesized by conventional techniques including automatedDNA synthesizers. Alternatively, PCR amplification of gene fragments canbe carried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (see,e.g., Ausubel et al., supra). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A nucleic acid encoding a polypeptide of the invention canbe cloned into such an expression vector such that the fusion moiety islinked in-frame to the polypeptide of the invention.

A signal sequence can be used to facilitate secretion and isolation ofthe secreted protein or other proteins of interest. Signal sequences aretypically characterized by a core of hydrophobic amino acids which aregenerally cleaved from the mature protein during secretion in one ormore cleavage events. Such signal peptides contain processing sites thatallow cleavage of the signal sequence from the mature proteins as theypass through the secretory pathway. Thus, the invention pertains to thedescribed polypeptides having a signal sequence, as well as topolypeptides from which the signal sequence has been proteolyticallycleaved (i.e., the cleavage products). In one embodiment, a nucleic acidsequence encoding a signal sequence can be operably linked in anexpression vector to a protein of interest, such as a protein which isordinarily not secreted or is otherwise difficult to isolate. The signalsequence directs secretion of the protein, such as from a eukaryotichost into which the expression vector is transformed, and the signalsequence is subsequently or concurrently cleaved. The protein can thenbe readily purified from the extracellular medium by art recognizedmethods. Alternatively, the signal sequence can be linked to the proteinof interest using a sequence which facilitates purification, such aswith a GST domain.

The present invention also pertains to variants of the polypeptidescorresponding to individual markers of the invention. Such variants havean altered amino acid sequence which can function as either agonists(mimetics) or as antagonists. Variants can be generated by mutagenesis,e.g., discrete point mutation or truncation. An agonist can retainsubstantially the same, or a subset, of the biological activities of thenaturally occurring form of the protein. An antagonist of a protein caninhibit one or more of the activities of the naturally occurring form ofthe protein by, for example, competitively binding to a downstream orupstream member of a cellular signaling cascade which includes theprotein of interest. Thus, specific biological effects can be elicitedby treatment with a variant of limited function. Treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein can have fewer side effects in asubject relative to treatment with the naturally occurring form of theprotein.

Variants of a protein of the invention which function as either agonists(mimetics) or as antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theprotein of the invention for agonist or antagonist activity. In oneembodiment, a variegated library of variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential protein sequences is expressible as individual polypeptides,or alternatively, as a set of larger fusion proteins (e.g., for phagedisplay). There are a variety of methods which can be used to producelibraries of potential variants of the polypeptides of the inventionfrom a degenerate oligonucleotide sequence. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang,1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem.53:323; Itakura et al., 1984, Science 198:1056; Ike et al., 1983 NucleicAcid Res. 1 1:477).

In addition, libraries of fragments of the coding sequence of apolypeptide corresponding to a marker of the invention can be used togenerate a variegated population of polypeptides for screening andsubsequent selection of variants. For example, a library of codingsequence fragments can be generated by treating a double stranded PCRfragment of the coding sequence of interest with a nuclease underconditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S I nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes amino terminal andinternal fragments of various sizes of the protein of interest.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein of the invention (Arkin and Yourvan, 1992, Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al., 1993, Protein Engineering6(3):327-331).

An isolated polypeptide corresponding to a marker of the invention, or afragment thereof, can be used as an immunogen to generate antibodiesusing standard techniques for polyclonal and monoclonal antibodypreparation. The full-length polypeptide or protein can be used or,alternatively, the invention provides antigenic peptide fragments foruse as immunogens. The antigenic peptide of a protein of the inventioncomprises at least 8 (preferably 10, 15, 20, or 30 or more) amino acidresidues of the amino acid sequence of one of the polypeptides of theinvention, and encompasses an epitope of the protein such that anantibody raised against the peptide forms a specific immune complex witha marker of the invention to which the protein corresponds. Preferredepitopes encompassed by the antigenic peptide are regions that arelocated on the surface of the protein, e.g., hydrophilic regions.Hydrophobicity sequence analysis, hydrophilicity sequence analysis, orsimilar analyses can be used to identify hydrophilic regions.

An immunogen typically is used to prepare antibodies by immunizing asuitable (i.e. immunocompetent) subject such as a rabbit, goat, mouse,or other mammal or vertebrate. An appropriate immunogenic preparationcan contain, for example, recombinantly-expressed orchemically-synthesized polypeptide. The preparation can further includean adjuvant, such as Freund's complete or incomplete adjuvant, or asimilar immunostimulatory agent.

Accordingly, another aspect of the invention pertains to antibodiesdirected against a polypeptide of the invention. The terms “antibody”and “antibody substance” as used interchangeably herein refer toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds an antigen, such as a polypeptideof the invention, e.g., an epitope of a polypeptide of the invention. Amolecule which specifically binds to a given polypeptide of theinvention is a molecule which binds the polypeptide, but does notsubstantially bind other molecules in a sample, e.g., a biologicalsample, which naturally contains the polypeptide. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)₂ fragments which can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies. The term “monoclonal antibody” or“monoclonal antibody composition”, as used herein, refers to apopulation of antibody molecules that contain only one species of anantigen binding site capable of immunoreacting with a particularepitope.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a polypeptide of the invention as an immunogen.Preferred polyclonal antibody compositions are ones that have beenselected for antibodies directed against a polypeptide or polypeptidesof the invention. Particularly preferred polyclonal antibodypreparations are ones that contain only antibodies directed against apolypeptide or polypeptides of the invention. Particularly preferredimmunogen compositions are those that contain no other human proteinssuch as, for example, immunogen compositions made using a non-human hostcell for recombinant expression of a polypeptide of the invention. Insuch a manner, the only human epitope or epitopes recognized by theresulting antibody compositions raised against this immunogen will bepresent as part of a polypeptide or polypeptides of the invention.

The antibody titer in the immunized subject can be monitored over timeby standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized polypeptide. If desired, the antibodymolecules can be harvested or isolated from the subject (e.g., from theblood or serum of the subject) and further purified by well-knowntechniques, such as protein A chromatography to obtain the IgG fraction.Alternatively, antibodies specific for a protein or polypeptide of theinvention can be selected or (e.g., partially purified) or purified by,e.g., affinity chromatography. For example, a recombinantly expressedand purified (or partially purified) protein of the invention isproduced as described herein, and covalently or non-covalently coupledto a solid support such as, for example, a chromatography column. Thecolumn can then be used to affinity purify antibodies specific for theproteins of the invention from a sample containing antibodies directedagainst a large number of different epitopes, thereby generating asubstantially purified antibody composition, i.e., one that issubstantially free of contaminating antibodies. By a substantiallypurified antibody composition is meant, in this context, that theantibody sample contains at most only 30% (by dry weight) ofcontaminating antibodies directed against epitopes other than those ofthe desired protein or polypeptide of the invention, and preferably atmost 20%, yet more preferably at most 10%, and most preferably at most5% (by dry weight) of the sample is contaminating antibodies. A purifiedantibody composition means that at least 99% of the antibodies in thecomposition are directed against the desired protein or polypeptide ofthe invention.

At an appropriate time after immunization, e.g., when the specificantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497, the human B cellhybridoma technique (see Kozbor et al., 1983, Immunol. Today 4:72), theEBV-hybridoma technique (see Cole et al., pp. 77-96 In MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., 1985) or triomatechniques. The technology for producing hybridomas is well known (seegenerally Current Protocols in Immunology, Coligan et al. ed., JohnWiley & Sons, New York, 1994). Hybridoma cells producing a monoclonalantibody of the invention are detected by screening the hybridomaculture supernatants for antibodies that bind the polypeptide ofinterest, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody directed against a polypeptide of the invention canbe identified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withthe polypeptide of interest. Kits for generating and screening phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally,examples of methods and reagents particularly amenable for use ingenerating and screening antibody display library can be found in, forexample, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCTPublication No. WO 91/17271; PCT Publication No. WO 92/20791; PCTPublication No. WO 92/15679; PCT Publication No. WO 93/01288; PCTPublication No. WO 92/01047; PCT Publication No. WO 92/09690; PCTPublication No. WO 90/02809; Fuchs et al (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J.12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. A chimeric antibody is a molecule in whichdifferent portions are derived from different animal species, such asthose having a variable region derived from a murine mAb and a humanimmunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which areincorporated herein by reference in their entirety.) Humanizedantibodies are antibody molecules from non-human species having one ormore complementarily determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated hereinby reference in its entirety.) Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in PCT Publication No. WO87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041 -1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Antibodies of the invention may be used as therapeutic agents intreating cancers. In a preferred embodiment, completely human antibodiesof the invention are used for therapeutic treatment of human cancerpatients, particularly those having an ovarian cancer. Such antibodiescan be produced, for example, using transgenic mice which are incapableof expressing endogenous immunoglobulin heavy and light chains genes,but which can express human heavy and light chain genes. The transgenicmice are immunized in the normal fashion with a selected antigen, e.g.,all or a portion of a polypeptide corresponding to a marker of theinvention. Monoclonal antibodies directed against the antigen can beobtained using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995) Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.), can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al., 1994, Bio/technology12:899-903).

An antibody directed against a polypeptide corresponding to a marker ofthe invention (e.g., a monoclonal antibody) can be used to isolate thepolypeptide by standard techniques, such as affinity chromatography orimmunoprecipitation. Moreover, such an antibody can be used to detectthe marker (e.g., in a cellular lysate or cell supernatant) in order toevaluate the level and pattern of expression of the marker. Theantibodies can also be used diagnostically to monitor protein levels intissues or body fluids (e.g. in an ovary-associated body fluid) as partof a clinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Further, an antibody (or fragment thereof) can be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, coichicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, .alpha.-interferon, .beta.-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

Accordingly, in one aspect, the invention provides substantiallypurified antibodies or fragments thereof, and non-human antibodies orfragments thereof, which antibodies or fragments specifically bind to apolypeptide comprising an amino acid sequence selected from the groupconsisting of the amino acid sequences of the present invention, anamino acid sequence encoded by the cDNA of the present invention, afragment of at least 15 amino acid residues of an amino acid sequence ofthe present invention, an amino acid sequence which is at least 95%identical to the amino acid sequence of the present invention (whereinthe percent identity is determined using the ALIGN program of the GCGsoftware package with a PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4) and an amino acid sequence whichis encoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule consisting of the nucleic acid molecules of the presentinvention, or a complement thereof, under conditions of hybridization of6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at 65° C. In variousembodiments, the substantially purified antibodies of the invention, orfragments thereof, can be human, non-human, chimeric and/or humanizedantibodies.

In another aspect, the invention provides non-human antibodies orfragments thereof, which antibodies or fragments specifically bind to apolypeptide comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of the present invention, anamino acid sequence encoded by the cDNA of the present invention, afragment of at least 15 amino acid residues of the amino acid sequenceof the present invention, an amino acid sequence which is at least 95%identical to the amino acid sequence of the present invention (whereinthe percent identity is determined using the ALIGN program of the GCGsoftware package with a PAM 120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4) and an amino acid sequence whichis encoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule consisting of the nucleic acid molecules of the presentinvention, or a complement thereof, under conditions of hybridization of6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at 65° C. Suchnon-human antibodies can be goat, mouse, sheep, horse, chicken, rabbit,or rat antibodies. Alternatively, the non-human antibodies of theinvention can be chimeric and/or humanized antibodies. In addition, thenon-human antibodies of the invention can be polyclonal antibodies ormonoclonal antibodies.

In still a further aspect, the invention provides monoclonal antibodiesor fragments thereof, which antibodies or fragments specifically bind toa polypeptide comprising an amino acid sequence selected from the groupconsisting of the amino acid sequences of the present invention, anamino acid sequence encoded by the cDNA of the present invention, afragment of at least 15 amino acid residues of an amino acid sequence ofthe present invention, an amino acid sequence which is at least 95%identical to an amino acid sequence of the present invention (whereinthe percent identity is determined using the ALIGN program of the GCGsoftware package with a PAMI 20 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4) and an amino acid sequence whichis encoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule consisting of the nucleic acid molecules of the presentinvention, or a complement thereof, under conditions of hybridization of6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at 65° C. Themonoclonal antibodies can be human, humanized, chimeric and/or non-humanantibodies.

The substantially purified antibodies or fragments thereof mayspecifically bind to a signal peptide, a secreted sequence, anextracellular domain, a transmembrane or a cytoplasmic domain orcytoplasmic membrane of a polypeptide of the invention. In aparticularly preferred embodiment, the substantially purified antibodiesor fragments thereof, the non-human antibodies or fragments thereof,and/or the monoclonal antibodies or fragments thereof, of the inventionspecifically bind to a secreted sequence or an extracellular domain ofthe amino acid sequences of the present invention.

Any of the antibodies of the invention can be conjugated to atherapeutic moiety or to a detectable substance. Non-limiting examplesof detectable substances that can be conjugated to the antibodies of theinvention are an enzyme, a prosthetic group, a fluorescent material, aluminescent material, a bioluminescent material, and a radioactivematerial.

The invention also provides a kit containing an antibody of theinvention conjugated to a detectable substance, and instructions foruse. Still another aspect of the invention is a pharmaceuticalcomposition comprising an antibody of the invention and apharmaceutically acceptable carrier. In preferred embodiments, thepharmaceutical composition contains an antibody of the invention, atherapeutic moiety, and a pharmaceutically acceptable carrier.

Still another aspect of the invention is a method of making an antibodythat specifically recognizes a polypeptide of the present invention, themethod comprising immunizing a mammal with a polypeptide. Thepolypeptide used as an immungen comprises an amino acid sequenceselected from the group consisting of the amino acid sequence of thepresent invention, an amino acid sequence encoded by the cDNA of thenucleic acid molecules of the present invention, a fragment of at least15 amino acid residues of the amino acid sequence of the presentinvention, an amino acid sequence which is at least 95% identical to theamino acid sequence of the present invention (wherein the percentidentity is determined using the ALIGN program of the GCG softwarepackage with a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4) and an amino acid sequence which is encoded by anucleic acid molecule which hybridizes to a nucleic acid moleculeconsisting of the nucleic acid molecules of the present invention, or acomplement thereof, under conditions of hybridization of 6×SSC at 45° C.and washing in 0.2×SSC, 0.1% SDS at 65° C.

After immunization, a sample is collected from the mammal that containsan antibody that specifically recognizes the polypeptide. Preferably,the polypeptide is recombinantly produced using a non-human host cell.Optionally, the antibodies can be further purified from the sample usingtechniques well known to those of skill in the art. The method canfurther comprise producing a monoclonal antibody-producing cell from thecells of the mammal. Optionally, antibodies are collected from theantibody-producing cell.

V. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptidecorresponding to a marker of the invention (or a portion of such apolypeptide). As used herein, the tenm “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, namely expressionvectors, are capable of directing the expression of genes to which theyare operably linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids (vectors).However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operably linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Methods in Enzymology: GeneExpression Technology vol. 185, Academic Press, San Diego, Calif.(1991). Regulatory sequences include those which direct constitutiveexpression of a nucleotide sequence in many types of host cell and thosewhich direct expression of the nucleotide sequence only in certain hostcells (e.g., tissue-specific regulatory sequences). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, andthe like. The expression vectors of the invention can be introduced intohost cells to thereby produce proteins or peptides, including fusionproteins or peptides, encoded by nucleic acids as described herein.

The recombinant expression vectors of the invention can be designed forexpression of a polypeptide corresponding to a marker of the inventionin prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells{using baculovirus expression vectors}, yeast cells or mammalian cells).Suitable host cells are discussed further in Goeddel, supra.Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studieret al., p. 60-89, In Gene Expression Technology: Methods in Enzymologyvol. 185, Academic Press, San Diego, Calif., 1991). Target geneexpression from the pTrc vector relies on host RNA polymerasetranscription from a hybrid trp-lac fusion promoter. Target geneexpression from the pET 11d vector relies on transcription from a T7gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase(T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3)or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene underthe transcriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, p. 119-128,In Gene Expression Technology: Methods in Enzymology vol. 185, AcademicPress, San Diego, Calif., 1990. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., 1992, Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjanand Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987,Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpPicZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.,1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers, 1989, Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840)and pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal., supra.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.,1987, Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton, 1988, Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) andimmunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen andBaltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al., 1985,Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss,1990, Science 249:374-379) and the α-fetoprotein promoter (Camper andTilghman, 1989, Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to the mRNA encoding a polypeptide of the invention.Regulatory sequences operably linked to a nucleic acid cloned in theantisense orientation can be chosen which direct the continuousexpression of the antisense RNA molecule in a variety of cell types, forinstance viral promoters and/or enhancers, or regulatory sequences canbe chosen which direct constitutive, tissue-specific or cell typespecific expression of antisense RNA. The antisense expression vectorcan be in the form of a recombinant plasmid, phagemid, or attenuatedvirus in which antisense nucleic acids are produced under the control ofa high efficiency regulatory region, the activity of which can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee Weintraub et al., 1986, Trends in Genetics, Vol. I (1).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell(e.g., insect cells, yeast or mammalian cells).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce a polypeptide corresponding to amarker of the invention. Accordingly, the invention further providesmethods for producing a polypeptide corresponding to a marker of theinvention using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding a polypeptide of the inventionhas been introduced) in a suitable medium such that the marker isproduced. In another embodiment, the method further comprises isolatingthe marker polypeptide from the medium or the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into which asequences encoding a polypeptide corresponding to a marker of theinvention have been introduced. Such host cells can then be used tocreate non-human transgenic animals in which exogenous sequencesencoding a marker protein of the invention have been introduced intotheir genome or homologous recombinant animals in which endogenousgene(s) encoding a polypeptide corresponding to a marker of theinvention sequences have been altered. Such animals are useful forstudying the function and/or activity of the polypeptide correspondingto the marker and for identifying and/or evaluating modulators ofpolypeptide activity. As used herein, a “transgenic animal” is anon-human animal, preferably a mammal, more preferably a rodent such asa rat or mouse, in which one or more of the cells of the animal includesa transgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, an “homologous recombinant animal” is anon-human animal, preferably a mammal, more preferably a mouse, in whichan endogenous gene has been altered by homologous recombination betweenthe endogenous gene and an exogenous DNA molecule introduced into a cellof the animal, e.g., an embryonic cell of the animal, prior todevelopment of the animal.

A transgenic animal of the invention can be created by introducing anucleic acid encoding a polypeptide corresponding to a marker of theinvention into the male pronuclei of a fertilized oocyte, e.g., bymicroinjection, retroviral infection, and allowing the oocyte to developin a pseudopregnant female foster animal. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the polypeptide of the invention toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan,Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986. Similar methods are used for production ofother transgenic animals. A transgenic founder animal can be identifiedbased upon the presence of the transgene in its genome and/or expressionof mRNA encoding the transgene in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying thetransgene can further be bred to other transgenic animals carrying othertransgenes.

To create an homologous recombinant animal, a vector is prepared whichcontains at least a portion of a gene encoding a polypeptidecorresponding to a marker of the invention into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the gene. In a preferred embodiment, the vector isdesigned such that, upon homologous recombination, the endogenous geneis functionally disrupted (i.e., no longer encodes a functional protein;also referred to as a “knock out” vector). Alternatively, the vector canbe designed such that, upon homologous recombination, the endogenousgene is mutated or otherwise altered but still encodes functionalprotein (e.g., the upstream regulatory region can be altered to therebyalter the expression of the endogenous protein). In the homologousrecombination vector, the altered portion of the gene is flanked at its5′ and 3′ ends by additional nucleic acid of the gene to allow forhomologous recombination to occur between the exogenous gene carried bythe vector and an endogenous gene in an embryonic stem cell. Theadditional flanking nucleic acid sequences are of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector (see, e.g., Thomas and Capecchi, 1987, Cell51:503 for a description of homologous recombination vectors). Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced gene has homologouslyrecombined with the endogenous gene are selected (see, e.g., Li et al.,1992, Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see, e.g., Bradley, Teratocarcinomas and Embryonic Stem Cells: APractical Approach, Robertson, Ed., IRL, Oxford, 1987, pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination vectors and homologous recombinantanimals are described further in Bradley (1991) Current Opinion inBio/Technology 2:823-829 and in PCT Publication NOS. WO 90/11354, WO91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.,1991, Science 251:1351-1355). If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO 97/07669.

VI. Pharmaceutical Compositions

The nucleic acid molecules, polypeptides, and antibodies (also referredto herein as “active compounds”) corresponding to a marker of theinvention can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the nucleicacid molecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

The invention includes methods for preparing pharmaceutical compositionsfor modulating the expression or activity of a polypeptide or nucleicacid corresponding to a marker of the invention. Such methods compriseformulating a pharmaceutically acceptable carrier with an agent whichmodulates expression or activity of a polypeptide or nucleic acidcorresponding to a marker of the invention. Such compositions canfurther include additional active agents. Thus, the invention furtherincludes methods for preparing a pharmaceutical composition byformulating a pharmaceutically acceptable carrier with an agent whichmodulates expression or activity of a polypeptide or nucleic acidcorresponding to a marker of the invention and one or more additionalactive compounds.

The invention also provides methods (also referred to herein as”screening assays”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., peptides, peptidomimetics, peptoids, smallmolecules or other drugs) which (a) bind to the marker, or (b) have amodulatory (e.g., stimulatory or inhibitory) effect on the activity ofthe marker or, more specifically, (c) have a modulatory effect on theinteractions of the marker with one or more of its natural substrates(e.g., peptide, protein, hormone, co-factor, or nucleic acid), or (d)have a modulatory effect on the expression of the marker. Such assaystypically comprise a reaction between the marker and one or more assaycomponents. The other components may be either the test compound itself,or a combination of test compound and a natural binding partner of themarker.

The test compounds of the present invention may be obtained from anyavailable source, including systematic libraries of natural and/orsynthetic compounds. Test compounds may also be obtained by any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann et al., 1994,J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam, 1997, AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/orspores, (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al, 1992,Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith, 1990,Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al,1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol.222:301-310; Ladner, supra.).

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a marker or biologicallyactive portion thereof. In another embodiment, the invention providesassays for screening candidate or test compounds which bind to a markeror biologically active portion thereof. Determining the ability of thetest compound to directly bind to a marker can be accomplished, forexample, by coupling the compound with a radioisotope or enzymatic labelsuch that binding of the compound to the marker can be determined bydetecting the labeled marker compound in a complex. For example,compounds (e.g., marker substrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemission or by scintillation counting.Alternatively, assay components can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product.

In another embodiment, the invention provides assays for screeningcandidate or test compounds which modulate the activity of a marker or abiologically active portion thereof. In all likelihood, the marker can,in vivo, interact with one or more molecules, such as but not limitedto, peptides, proteins, hormones, cofactors and nucleic acids. For thepurposes of this discussion, such cellular and extracellular moleculesare referred to herein as “binding partners” or marker “substrate”.

One necessary embodiment of the invention in order to facilitate suchscreening is the use of the marker to identify its natural in vivobinding partners. There are many ways to accomplish this which are knownto one skilled in the art. One example is the use of the marker proteinas “bait protein” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al, 1993, Cell 72:223-232;Madura et al, 1993, J. Biol. Chem. 268:12046-12054; Bartel et al, 1993,Biotechniques 14:920-924; Iwabuchi et al, 1993 Oncogene 8:1693-1696;Brent WO94/10300) in order to identify other proteins which bind to orinteract with the marker (binding partners) and, therefore, are possiblyinvolved in the natural function of the marker. Such marker bindingpartners are also likely to be involved in the propagation of signals bythe marker or downstream elements of a marker-mediated signalingpathway. Alternatively, such marker binding partners may also be foundto be inhibitors of the marker.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that encodes a marker proteinfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a marker-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be readily detected and cell colonies containingthe functional transcription factor can be isolated and used to obtainthe cloned gene which encodes the protein which interacts with themarker protein.

In a further embodiment, assays may be devised through the use of theinvention for the purpose of identifying compounds which modulate (e.g.,affect either positively or negatively) interactions between a markerand its substrates and/or binding partners. Such compounds can include,but are not limited to, molecules such as antibodies, peptides,hormones, oligonucleotides, nucleic acids, and analogs thereof. Suchcompounds may also be obtained from any available source, includingsystematic libraries of natural and/or synthetic compounds. Thepreferred assay components for use in this embodiment is an ovariancancer marker identified herein, the known binding partner and/orsubstrate of same, and the test compound. Test compounds can be suppliedfrom any source.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the marker and its bindingpartner involves preparing a reaction mixture containing the marker andits binding partner under conditions and for a time sufficient to allowthe two products to interact and bind, thus forming a complex. In orderto test an agent for inhibitory activity, the reaction mixture isprepared in the presence and absence of the test compound. The testcompound can be initially included in the reaction mixture, or can beadded at a time subsequent to the addition of the marker and its bindingpartner. Control reaction mixtures are incubated without the testcompound or with a placebo. The formation of any complexes between themarker and its binding partner is then detected. The formation of acomplex in the control reaction, but less or no such formation in thereaction mixture containing the test compound, indicates that thecompound interferes with the interaction of the marker and its bindingpartner. Conversely, the formation of more complex in the presence ofcompound than in the control reaction indicates that the compound mayenhance interaction of the marker and its binding partner.

The assay for compounds that interfere with the interaction of themarker with its binding partner may be conducted in a heterogeneous orhomogeneous format. Heterogeneous assays involve anchoring either themarker or its binding partner onto a solid phase and detecting complexesanchored to the solid phase at the end of the reaction. In homogeneousassays, the entire reaction is carried out in a liquid phase. In eitherapproach, the order of addition of reactants can be varied to obtaindifferent information about the compounds being tested. For example,test compounds that interfere with the interaction between the markersand the binding partners (e.g., by competition) can be identified byconducting the reaction in the presence of the test substance, i.e., byadding the test substance to the reaction mixture prior to orsimultaneously with the marker and its interactive binding partner.Alternatively, test compounds that disrupt preformed complexes, e.g.,compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are briefly described below.

In a heterogeneous assay system, either the marker or its bindingpartner is anchored onto a solid surface or matrix, while the othercorresponding non-anchored component may be labeled, either directly orindirectly. In practice, microtitre plates are often utilized for thisapproach. The anchored species can be immobilized by a number ofmethods, either non-covalent or covalent, that are typically well knownto one who practices the art. Non-covalent attachment can often beaccomplished simply by coating the solid surface with a solution of themarker or its binding partner and drying. Alternatively, an immobilizedantibody specific for the assay component to be anchored can be used forthis purpose. Such surfaces can often be prepared in advance and stored.

In related embodiments, a fusion protein can be provided which adds adomain that allows one or both of the assay components to be anchored toa matrix. For example, glutathione-S-transferase/marker fusion proteinsor glutathione-S-transferase/binding partner can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedmarker or its binding partner, and the mixture incubated underconditions conducive to complex formation (e.g., physiologicalconditions). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound assay components, the immobilizedcomplex assessed either directly or indirectly, for example, asdescribed above. Alternatively, the complexes can be dissociated fromthe matrix, and the level of marker binding or activity determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a markeror a marker binding partner can be immobilized utilizing conjugation ofbiotin and streptavidin. Biotinylated marker protein or target moleculescan be prepared from biotin-NHS (N-hydroxy-succinimide) using techniquesknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). In certain embodiments, theprotein-immobilized surfaces can be prepared in advance and stored.

In order to conduct the assay, the corresponding partner of theimmobilized assay component is exposed to the coated surface with orwithout the test compound. After the reaction is complete, unreactedassay components are removed (e.g., by washing) and any complexes formedwill remain immobilized on the solid surface. The detection of complexesanchored on the solid surface can be accomplished in a number of ways.Where the non-immobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the initially non-immobilizedspecies (the antibody, in turn, can be directly labeled or indirectlylabeled with, e.g., a labeled anti-Ig antibody). Depending upon theorder of addition of reaction components, test compounds which modulate(inhibit or enhance) complex formation or which disrupt preformedcomplexes can be detected.

In an alternate embodiment of the invention, a homogeneous assay may beused. This is typically a reaction, analogous to those mentioned above,which is conducted in a liquid phase in the presence or absence of thetest compound. The formed complexes are then separated from unreactedcomponents, and the amount of complex formed is determined. As mentionedfor heterogeneous assay systems, the order of addition of reactants tothe liquid phase can yield information about which test compoundsmodulate (inhibit or enhance) complex formation and which disruptpreformed complexes.

In such a homogeneous assay, the reaction products may be separated fromunreacted assay components by any of a number of standard techniques,including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, complexes of molecules may be separated from uncomplexedmolecules through a series of centrifugal steps, due to the differentsedimentation equilibria of complexes based on their different sizes anddensities (see, for example, Rivas, G., and Minton, A. P., TrendsBiochem Sci August 1993;18(8):284-7). Standard chromatographictechniques may also be utilized to separate complexed molecules fromuncomplexed ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format, for example, the relativelylarger complex may be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of thecomplex as compared to the uncomplexed molecules may be exploited todifferentially separate the complex from the remaining individualreactants, for example through the use of ion-exchange chromatographyresins. Such resins and chromatographic techniques are well known to oneskilled in the art (see, e.g., Heegaard, 1998, J Mol. Recognit. 11:141-148; Hage and Tweed, 1997, J. Chromatogr. B. Biomed. Sci. Appl.,699:499-525). Gel electrophoresis may also be employed to separatecomplexed molecules from unbound species (see, e.g., Ausubel et al(eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, NewYork. 1999). In this technique, protein or nucleic acid complexes areseparated based on size or charge, for example. In order to maintain thebinding interaction during the electrophoretic process, nondenaturinggels in the absence of reducing agent are typically preferred, butconditions appropriate to the particular interactants will be well knownto one skilled in the art. Immunoprecipitation is another commontechnique utilized for the isolation of a protein-protein complex fromsolution (see, e.g., Ausubel et al (eds.), In: Current Protocols inMolecular Biology, J. Wiley & Sons, New York. 1999). In this technique,all proteins binding to an antibody specific to one of the bindingmolecules are precipitated from solution by conjugating the antibody toa polymer bead that may be readily collected by centrifugation. Thebound assay components are released from the beads (through a specificproteolysis event or other technique well known in the art which willnot disturb the protein-protein interaction in the complex), and asecond immunoprecipitation step is performed, this time utilizingantibodies specific for the correspondingly different interacting assaycomponent. In this manner, only formed complexes should remain attachedto the beads. Variations in complex formation in both the presence andthe absence of a test compound can be compared, thus offeringinformation about the ability of the compound to modulate interactionsbetween the marker and its binding partner.

Also within the scope of the present invention are methods for directdetection of interactions between the marker and its natural bindingpartner and/or a test compound in a homogeneous or heterogeneous assaysystem without further sample manipulation. For example, the techniqueof fluorescence energy transfer may be utilized (see, e.g., Lakowicz etal, U.S. Pat. No. 5,631,169; Stavrianopoulos et al, U.S. Pat. No.4,868,103). Generally, this technique involves the addition of afluorophore label on a first ‘donor’ molecule (e.g., marker or testcompound) such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule (e.g., marker or testcompound), which in turn is able to fluoresce due to the absorbedenergy. Alternately, the ‘donor’ protein molecule may simply utilize thenatural fluorescent energy of tryptophan residues. Labels are chosenthat emit different wavelengths of light, such that the ‘acceptor’molecule label may be differentiated from that of the ‘donor’. Since theefficiency of energy transfer between the labels is related to thedistance separating the molecules, spatial relationships between themolecules can be assessed. In a situation in which binding occursbetween the molecules, the fluorescent emission of the ‘acceptor’molecule label in the assay should be maximal. An FET binding event canbe conveniently measured through standard fluorometric detection meanswell known in the art (e.g., using a fluorimeter). A test substancewhich either enhances or hinders participation of one of the species inthe preformed complex will result in the generation of a signal variantto that of background. In this way, test substances that modulateinteractions between a marker and its binding partner can be identifiedin controlled assays.

In another embodiment, modulators of marker expression are identified ina method wherein a cell is contacted with a candidate compound and theexpression of mRNA or protein, corresponding to a marker in the cell, isdetermined. The level of expression of mRNA or protein in the presenceof the candidate compound is compared to the level of expression of mRNAor protein in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of marker expressionbased on this comparison. For example, when expression of marker mRNA orprotein is greater (statistically significantly greater) in the presenceof the candidate compound than in its absence, the candidate compound isidentified as a stimulator of marker mRNA or protein expression.Conversely, when expression of marker mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of marker mRNA or protein expression. The level of marker mRNAor protein expression in the cells can be determined by methodsdescribed herein for detecting marker mRNA or protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a marker protein can be furtherconfirmed in vivo, e.g., in a whole animal model for cellulartransformation and/or tumorigenesis.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., an marker modulating agent, an antisense markernucleic acid molecule, an marker-specific antibody, or an marker-bindingpartner) can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

It is understood that appropriate doses of small molecule agents andprotein or polypeptide agents depends upon a number of factors withinthe knowledge of the ordinarily skilled physician, veterinarian, orresearcher. The dose(s) of these agents will vary, for example,depending upon the identity, size, and condition of the subject orsample being treated, further depending upon the route by which thecomposition is to be administered, if applicable, and the effect whichthe practitioner desires the agent to have upon the nucleic acid orpolypeptide of the invention. Exemplary doses of a small moleculeinclude milligram or microgram amounts per kilogram of subject or sampleweight (e.g. about 1 microgram per kilogram to about 500 milligrams perkilogram, about 100 micrograms per kilogram to about 5 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram). Exemplary doses of a protein or polypeptide include gram,milligram or microgram amounts per kilogram of subject or sample weight(e.g. about 1 microgram per kilogram to about 5 grams per kilogram,about 100 micrograms per kilogram to about 500 milligrams per kilogram,or about I milligram per kilogram to about 50 milligrams per kilogram).It is furthermore understood that appropriate doses of one of theseagents depend upon the potency of the agent with respect to theexpression or activity to be modulated. Such appropriate doses can bedetermined using the assays described herein. When one or more of theseagents is to be administered to an animal (e.g. a human) in order tomodulate expression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher can, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific agent employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediamine-tetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a polypeptide or antibody) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium, and thenincorporating the required other ingredients from those enumeratedabove. In the case of sterile powders for the preparation of sterileinjectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches, and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes having monoclonal antibodies incorporated thereinor thereon) can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of bodyweight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act inthe brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into theovarian epithelium). A method for lipidation of antibodies is describedby Cruikshank et al. (1997) J. Acquired Immune Deficiency Syndromes andHuman Retrovirology 14:193.

The nucleic acid molecules corresponding to a marker of the inventioncan be inserted into vectors and used as gene therapy vectors. Genetherapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (U.S. Pat. No. 5,328,470),or by stereotactic injection (see, e.g., Chen et al., 1994, Proc. Natl.Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the genetherapy vector can include the gene therapy vector in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery vector can be produced intact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can include one ormore cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

VII. Monitoring the Effectiveness of an Anti-Cancer Agent

As discussed above, the identified sensitivity and resistance genes canalso be used as markers to assess whether a tumor has become refractoryto an ongoing treatment (e.g., a chemotherapeutic treatment). When atumor is no longer responding to a treatment the expression profile ofthe tumor cells will change: the level of expression of one or more ofthe sensitivity genes will be reduced and/or the level of expression ofone or more of the resistance genes will increase.

In such a use, the invention provides methods for determining whether ananti-cancer treatment should be continued in a cancer patient,comprising the steps of:

a) obtaining two or more samples of cancer cells from a patientundergoing anti-cancer therapy;

b) determining the level of expression of one or more markers selectedfrom the group consisting of the markers of Tables 1-4 in the sampleexposed to the agent and in a sample of cancer cells that is not exposedto the agent; and

c) discontinuing or altering treatment when the expression of one ormore sensitivity genes decreases and/or when the expression of one ormore resistance genes increases.

As used herein, a patient refers to any subject undergoing treatment forcancer. The preferred subject will be a human patient undergoingchemotherapy treatment.

This embodiment of the present invention relies on comparing two or moresamples obtained from a patient undergoing anti-cancer treatment. Ingeneral, it is preferable to obtain a first sample from the patientprior to beginning therapy and one or more samples during treatment. Insuch a use, a baseline of expression prior to therapy is determined andthen changes in the baseline state of expression is monitored during thecourse of therapy. Alternatively, two or more successive samplesobtained during treatment can be used without the need of apre-treatment baseline sample. In such a use, the first sample obtainedfrom the subject is used as a baseline for determining whether theexpression of a particular gene is increasing or decreasing.

In general, when monitoring the effectiveness of a therapeutictreatment, two or more samples from the patient are examined.Preferably, three or more successively obtained samples are used,including at least one pretreatment sample.

VIII. Detection Assays

An exemplary method for detecting the presence or absence of apolypeptide or nucleic acid corresponding to a marker of the inventionin a biological sample involves obtaining a biological sample (e.g. anovarian tumor sample) from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting the polypeptideor nucleic acid (e.g., mRNA, genomic DNA, or cDNA). The detectionmethods of the invention can thus be used to detect mRNA, protein, cDNA,or genomic DNA, for example, in a biological sample in vitro as well asin vivo. For example, in vitro techniques for detection of mRNA includeNorthern hybridizations and in situ hybridizations. In vitro techniquesfor detection of a polypeptide corresponding to a marker of theinvention include enzyme linked immunosorbent assays (ELISAs), Westernblots, immunoprecipitations and immunofluorescence. In vitro techniquesfor detection of genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of a polypeptidecorresponding to a marker of the invention include introducing into asubject a labeled antibody directed against the polypeptide. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques.

A general principle of such diagnostic and prognostic assays involvespreparing a sample or reaction mixture that may contain a marker, and aprobe, under appropriate conditions and for a time sufficient to allowthe marker and probe to interact and bind, thus forming a complex thatcan be removed and/or detected in the reaction mixture. These assays canbe conducted in a variety of ways.

For example, one method to conduct such an assay would involve anchoringthe marker or probe onto a solid phase support, also referred to as asubstrate, and detecting target marker/probe complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, a sample from a subject, which is to be assayed for presenceand/or concentration of marker, can be anchored onto a carrier or solidphase support. In another embodiment, the reverse situation is possible,in which the probe can be anchored to a solid phase and a sample from asubject can be allowed to react as an unanchored component of the assay.

There are many established methods for anchoring assay components to asolid phase. These include, without limitation, marker or probemolecules which are immobilized through conjugation of biotin andstreptavidin. Such biotinylated assay components can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). In certain embodiments, the surfaces with immobilized assaycomponents can be prepared in advance and stored.

Other suitable carriers or solid phase supports for such assays includeany material capable of binding the class of molecule to which themarker or probe belongs. Well-known supports or carriers include, butare not limited to, glass, polystyrene, nylon, polypropylene, nylon,polyethylene, dextran, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite.

In order to conduct assays with the above mentioned approaches, thenon-immobilized component is added to the solid phase upon which thesecond component is anchored. After the reaction is complete,uncomplexed components may be removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized uponthe solid phase. The detection of marker/probe complexes anchored to thesolid phase can be accomplished in a number of methods outlined herein.

In a preferred embodiment, the probe, when it is the unanchored assaycomponent, can be labeled for the purpose of detection and readout ofthe assay, either directly or indirectly, with detectable labelsdiscussed herein and which are well-known to one skilled in the art.

It is also possible to directly detect marker/probe complex formationwithout further manipulation or labeling of either component (marker orprobe), for example by utilizing the technique of fluorescence energytransfer (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169;Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore labelon the first, ‘donor’ molecule is selected such that, upon excitationwith incident light of appropriate wavelength, its emitted fluorescentenergy will be absorbed by a fluorescent label on a second ‘acceptor’molecule, which in turn is able to fluoresce due to the absorbed energy.Alternately, the ‘donor’ protein molecule may simply utilize the naturalfluorescent energy of tryptophan residues. Labels are chosen that emitdifferent wavelengths of light, such that the ‘acceptor’ molecule labelmay be differentiated from that of the ‘donor’. Since the efficiency ofenergy transfer between the labels is related to the distance separatingthe molecules, spatial relationships between the molecules can beassessed. In a situation in which binding occurs between the molecules,the fluorescent emission of the ‘acceptor’ molecule label in the assayshould be maximal. An FET binding event can be conveniently measuredthrough standard fluorometric detection means well known in the art(e.g., using a fluorimeter).

In another embodiment, determination of the ability of a probe torecognize a marker can be accomplished without labeling either assaycomponent (probe or marker) by utilizing a technology such as real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. andUrbaniczky, C., 1991, Anal. Chem. 63:2338-2345 and Szabo et al., 1995,Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” or “surfaceplasmon resonance” is a technology for studying biospecific interactionsin real time, without labeling any of the interactants (e.g., BIAcore).Changes in the mass at the binding surface (indicative of a bindingevent) result in alterations of the refractive index of light near thesurface (the optical phenomenon of surface plasmon resonance (SPR)),resulting in a detectable signal which can be used as an indication ofreal-time reactions between biological molecules.

Alternatively, in another embodiment, analogous diagnostic andprognostic assays can be conducted with marker and probe as solutes in aliquid phase. In such an assay, the complexed marker and probe areseparated from uncomplexed components by any of a number of standardtechniques, including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, marker/probe complexes may be separated from uncomplexedassay components through a series of centrifugal steps, due to thedifferent sedimentation equilibria of complexes based on their differentsizes and densities (see, for example, Rivas, G., and Minton, A. P.,1993, Trends Biochem Sci. 18(8):284-7). Standard chromatographictechniques may also be utilized to separate complexed molecules fromuncomplexed ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format, for example, the relativelylarger complex may be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of themarker/probe complex as compared to the uncomplexed components may beexploited to differentiate the complex from uncomplexed components, forexample through the utilization of ion-exchange chromatography resins.Such resins and chromatographic techniques are well known to one skilledin the art (see, e.g., Heegaard, N. H., 1998, J. Mol. Recognit. Winter11(1-6):141-8; Hage, D. S., and Tweed, S. A. J. Chromatogr B Biomed SciAppl Oct. 10, 1997;699(1-2):499-525). Gel electrophoresis may also beemployed to separate complexed assay components from unbound components(see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology,John Wiley & Sons, New York, 1987-1999). In this technique, protein ornucleic acid complexes are separated based on size or charge, forexample. In order to maintain the binding interaction during theelectrophoretic process, non-denaturing gel matrix materials andconditions in the absence of reducing agent are typically preferred.Appropriate conditions to the particular assay and components thereofwill be well known to one skilled in the art.

In a particular embodiment, the level of mRNA corresponding to themarker can be determined both by in situ and by in vitro formats in abiological sample using methods known in the art. The term “biologicalsample” is intended to include tissues, cells, biological fluids andisolates thereof, isolated from a subject, as well as tissues, cells andfluids present within a subject. Many expression detection methods useisolated RNA. For in vitro methods, any RNA isolation technique thatdoes not select against the isolation of mRNA can be utilized for thepurification of RNA from ovarian cells (see, e.g., Ausubel et al., ed.,Current Protocols in Molecular Biology, John Wiley & Sons, New York1987-1999). Additionally, large numbers of tissue samples can readily beprocessed using techniques well known to those of skill in the art, suchas, for example, the single-step RNA isolation process of Chomczynski(1989, U.S. Pat. No. 4,843,155).

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One preferreddiagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length cDNA, or a portionthereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250or 500 nucleotides in length and sufficient to specifically hybridizeunder stringent conditions to a mRNA or genomic DNA encoding a marker ofthe present invention. Other suitable probes for use in the diagnosticassays of the invention are described herein. Hybridization of an mRNAwith the probe indicates that the marker in question is being expressed.

In one format, the mRNA is immobilized on a solid surface and contactedwith a probe, for example by running the isolated mRNA on an agarose geland transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probe(s) are immobilizedon a solid surface and the mRNA is contacted with the probe(s), forexample, in an Affymetrix gene chip array. A skilled artisan can readilyadapt known mRNA detection methods for use in detecting the level ofmRNA encoded by the markers of the present invention.

An alternative method for determining the level of mRNA corresponding toa marker of the present invention in a sample involves the process ofnucleic acid amplification, e.g., by rtPCR (the experimental embodimentset forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chainreaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193), selfsustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad.Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh etal., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi et al., 1988, Bio/Technology 6:1197), rolling circlereplication (Lizardi et al., U.S. Pat. No. 5,854,033) or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers. As used herein, amplification primers are defined as being apair of nucleic acid molecules that can anneal to 5′ or 3′ regions of agene (plus and minus strands, respectively, or vice-versa) and contain ashort region in between. In general, amplification primers are fromabout 10 to 30 nucleotides in length and flank a region from about 50 to200 nucleotides in length. Under appropriate conditions and withappropriate reagents, such primers permit the amplification of a nucleicacid molecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the ovariancells prior to detection. In such methods, a cell or tissue sample isprepared/processed using known histological methods. The sample is thenimmobilized on a support, typically a glass slide, and then contactedwith a probe that can hybridize to mRNA that encodes the marker.

As an alternative to making determinations based on the absoluteexpression level of the marker, determinations may be based on thenormalized expression level of the marker. Expression levels arenormalized by correcting the absolute expression level of a marker bycomparing its expression to the expression of a gene that is not amarker, e.g., a housekeeping gene that is constitutively expressed.Suitable genes for normalization include housekeeping genes such as theactin gene, or epithelial cell-specific genes. This normalization allowsthe comparison of the expression level in one sample, e.g., a patientsample, to another sample, e.g., a non-ovarian cancer sample, or betweensamples from different sources.

Alternatively, the expression level can be provided as a relativeexpression level. To determine a relative expression level of a marker,the level of expression of the marker is determined for 10 or moresamples of normal versus cancer cell isolates, preferably 50 or moresamples, prior to the determination of the expression level for thesample in question. The mean expression level of each of the genesassayed in the larger number of samples is determined and this is usedas a baseline expression level for the marker. The expression level ofthe marker determined for the test sample (absolute level of expression)is then divided by the mean expression value obtained for that marker.This provides a relative expression level.

In another embodiment of the present invention, a polypeptidecorresponding to a marker is detected. A preferred agent for detecting apolypeptide of the invention is an antibody capable of binding to apolypeptide corresponding to a marker of the invention, preferably anantibody with a detectable label. Antibodies can be polyclonal, or morepreferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,Fab or F(ab′)₂) can be used. The term “labeled”, with regard to theprobe or antibody, is intended to encompass direct labeling of the probeor antibody by coupling (i.e., physically linking) a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with another reagent that is directlylabeled. Examples of indirect labeling include detection of a primaryantibody using a fluorescently labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently labeled streptavidin.

A variety of formats can be employed to determine whether a samplecontains a protein that binds to a given antibody. Examples of suchformats include, but are not limited to, enzyme immunoassay (EIA),radioimmunoassay (RIA), Western blot analysis and enzyme linkedimmunoabsorbant assay (ELISA). A skilled artisan can readily adapt knownprotein/antibody detection methods for use in determining whetherovarian cells express a marker of the present invention.

In one format, antibodies, or antibody fragments, can be used in methodssuch as Western blots or immunofluorescence techniques to detect theexpressed proteins. In such uses, it is generally preferable toimmobilize either the antibody or proteins on a solid support. Suitablesolid phase supports or carriers include any support capable of bindingan antigen or an antibody. Well-known supports or carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite.

One skilled in the art will know many other suitable carriers forbinding antibody or antigen, and will be able to adapt such support foruse with the present invention. For example, protein isolated fromovarian cells can be run on a polyacrylamide gel electrophoresis andimmobilized onto a solid phase support such as nitrocellulose. Thesupport can then be washed with suitable buffers followed by treatmentwith the detectably labeled antibody. The solid phase support can thenbe washed with the buffer a second time to remove unbound antibody. Theamount of bound label on the solid support can then be detected byconventional means.

The invention also encompasses kits for detecting the presence of apolypeptide or nucleic acid corresponding to a marker of the inventionin a biological sample (e.g. an ovary-associated body fluid such as aurine sample). Such kits can be used to determine if a subject issuffering from or is at increased risk of developing ovarian cancer. Forexample, the kit can comprise a labeled compound or agent capable ofdetecting a polypeptide or an mRNA encoding a polypeptide correspondingto a marker of the invention in a biological sample and means fordetermining the amount of the polypeptide or mRNA in the sample (e.g.,an antibody which binds the polypeptide or an oligonucleotide probewhich binds to DNA or mRNA encoding the polypeptide). Kits can alsoinclude instructions for interpreting the results obtained using thekit.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to apolypeptide corresponding to a marker of the invention; and, optionally,(2) a second, different antibody which binds to either the polypeptideor the first antibody and is conjugated to a detectable label.

For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptidecorresponding to a marker of the invention or (2) a pair of primersuseful for amplifying a nucleic acid molecule corresponding to a markerof the invention. The kit can also comprise, e.g., a buffering agent, apreservative, or a protein stabilizing agent. The kit can furthercomprise components necessary for detecting the detectable label (e.g.,an enzyme or a substrate). The kit can also contain a control sample ora series of control samples which can be assayed and compared to thetest sample. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package, along with instructions for interpreting the results ofthe assays performed using the kit.

IX. Electronic Apparatus Readable Media and Arrays

Electronic apparatus readable media comprising a marker of the presentinvention is also provided. As used herein, “electronic apparatusreadable media” refers to any suitable medium for storing, holding orcontaining data or information that can be read and accessed directly byan electronic apparatus. Such media can include, but are not limited to:magnetic storage media, such as floppy discs, hard disc storage medium,and magnetic tape; optical storage media such as compact disc;electronic storage media such as RAM, ROM, EPROM, EEPROM and the like;general hard disks and hybrids of these categories such asmagnetic/optical storage media. The medium is adapted or configured forhaving recorded thereon a marker of the present invention.

As used herein, the term “electronic apparatus” is intended to includeany suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as a personal digital assistants(PDAs), cellular phone, pager and the like; and local and distributedprocessing systems.

As used herein, “recorded” refers to a process for storing or encodinginformation on the electronic apparatus readable medium. Those skilledin the art can readily adopt any of the presently known methods forrecording information on known media to generate manufactures comprisingthe markers of the present invention.

A variety of software programs and formats can be used to store themarker information of the present invention on the electronic apparatusreadable medium. For example, the nucleic acid sequence corresponding tothe markers can be represented in a word processing text file, formattedin commercially-available software such as WordPerfect and MicroSoftWord, or represented in the form of an ASCII file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like, as well as inother forms. Any number of dataprocessor structuring formats (e.g., textfile or database) may be employed in order to obtain or create a mediumhaving recorded thereon the markers of the present invention.

By providing the markers of the invention in readable form, one canroutinely access the marker sequence information for a variety ofpurposes. For example, one skilled in the art can use the nucleotide oramino acid sequences of the present invention in readable form tocompare a target sequence or target structural motif with the sequenceinformation stored within the data storage means. Search means are usedto identify fragments or regions of the sequences of the invention whichmatch a particular target sequence or target motif.

The invention also includes an array comprising a marker of the presentinvention. The array can be used to assay expression of one or moregenes in the array. In one embodiment, the array can be used to assaygene expression in a tissue to ascertain tissue specificity of genes inthe array. In this manner, up to about 36,000 genes can besimultaneously assayed for expression. This allows a profile to bedeveloped showing a battery of genes specifically expressed in one ormore tissues.

In addition to such qualitative determination, the invention allows thequantitation of gene expression. Thus, not only tissue specificity, butalso the level of expression of a battery of genes in the tissue isascertainable. Thus, genes can be grouped on the basis of their tissueexpression per se and level of expression in that tissue. This isuseful, for example, in ascertaining the relationship of gene expressionbetween or among tissues. Thus, one tissue can be perturbed and theeffect on gene expression in a second tissue can be determined. In thiscontext, the effect of one cell type on another cell type in response toa biological stimulus can be determined. Such a determination is useful,for example, to know the effect of cell-cell interaction at the level ofgene expression. If an agent is administered therapeutically to treatone cell type but has an undesirable effect on another cell type, theinvention provides an assay to determine the molecular basis of theundesirable effect and thus provides the opportunity to co-administer acounteracting agent or otherwise treat the undesired effect. Similarly,even within a single cell type, undesirable biological effects can bedetermined at the molecular level. Thus, the effects of an agent onexpression of other than the target gene can be ascertained andcounteracted.

In another embodiment, the array can be used to monitor the time courseof expression of one or more genes in the array.

The array is also useful for ascertaining the effect of the expressionof a gene on the expression of other genes in the same cell or indifferent cells. This provides, for example, for a selection ofalternate molecular targets for therapeutic intervention if the ultimateor downstream target cannot be regulated.

The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes that could serve as a molecular target fordiagnosis or therapeutic intervention.

SPECIFIC EXAMPLES

A. Identification Of Sensitivity And Resistant Markers

Tumor tissue from patients that were deemed sensitive to combinationTAXOL and cisplatin therapy (i.e., the patient was alive for 5.5 yearsafter treatment), and tumor tissue from patients that were deemedresistant to combination TAXOL and cisplatin therapy (i.e., the patientlived for either 10 or 15 months after treatment) were used to createsubtracted libraries. Subtracted libraries were generated using a PCRbased method that allows the isolation of clones expressed at higherlevels in one population of mRNA (tester) compared to another population(driver). Both tester and driver mRNA populations are converted intocDNA by reverse transcription, and then PCR amplified using the SMARTPCR kit from Clontech. Tester and driver cDNAs are then hybridized usingthe PCR-Select cDNA subtraction kit from Clontech. This techniqueresults in both subtraction and normalization, which is an equalizationof copy number of low-abundance and high-abundance sequences.

The “tester” source for one of the subtracted libraries was comprised ofcDNA generated from TAXOL-resistant tumor RNA Ov738. The “driver” sourcefor this library was comprised of cDNA generated from TAXOL-sensitivetumor RNA Ov 353. A second subtracted library was constructed from thesame cDNA samples, where the “tester” source was comprised of Ov 353,and the “driver”source was Ov 738. A third subtracted library wasconstructed wherein the “driver” source comprised of cDNA from the RNAof a TAXOL-resistant tumor sample Ov 844, and the “tester” source fromOv 353, a TAXOL-sensitive tumor RNA sample. A fourth subtracted librarywas constructed wherein the “driver” source comprised of Ov 353 and the“tester” source comprised of Ov 844.

Twelve-thousand clones were randomly picked from each library, PCRamplified and used for transcript profiling. Eleven ovarian tumorsamples (five sensitive and six resistant) were used for hybridizationagainst the nylon arrays. In particular, RNA was isolated from theovarian tumors using a Quagen RNeasy kit according to the manufacturersdirections. Probes for transcriptional profiling were generated byreverse transcribing the RNA into cDNA with Superscript II Reversetranscriptase, done in the presence of x³³P-dCTP. Transcriptionalprofiling was then performed using the radio labeled cDNA probe byhybridizing the probe to nylon filter arrays on which were spottedthe-twelve-thousand target cDNAs from the subtracted libraries. Thehybridizing of the specific cDNA probes to the target cDNAs was done for18 hours at 65° C. in the presence of Cot1 and Salmon sperm DNA to blocknon-specific binding. The filters were then washed once with 4% SDS-lowstringency wash buffer and twice with 1% SDS-high stringency washbuffer. After drying the filters they were placed on a Fujiphospho-imager screen for 48 hours. The image was then read on a Fujiphosphoimager, the intensity of the cDNA probe bound to target cDNAdigitized using Grid Guru and AIMZOO software packages.

The markers of Tables 1-4 were differentially expressed and chosen fromthe twelve thousand by statistical methods, including the use ofstatistical algorithms.

B. Therapeutic Agents

The markers of the present invention are shown to be sensitive orresistant to TAXOL. TAXOL is a chemical compound within a family oftaxane compounds which are art-recognized as being a family of relatedcompounds. The language “taxane compound” is intended to include TAXOL,compounds which are structurally similar to TAXOL and/or analogs ofTAXOL. The language “taxane compound” can also include “mimics”.“Mimics” is intended to include compounds which may not be structurallysimilar to TAXOL but mimic the therapeutic activity of TAXOL orstructurally similar taxane compounds in vivo. The taxane compounds ofthis invention are those compounds which are useful for inhibiting tumorgrowth in subjects (patients). The term taxane compound also is intendedto include pharmaceutically acceptable salts of the compounds. Taxanecompounds have previously been described in U.S. Pat. Nos. 5,641,803,5,665,671, 5,380,751, 5,728,687, 5,415,869, 5,407,683, 5,399,363,5,424,073, 5,157,049, 5,773,464, 5,821,263, 5,840,929, 4,814,470,5,438,072, 5,403,858, 4,960,790, 5,433,364, 4,942,184, 5,362,831,5,705,503, and 5,278,324, all of which are expressly incorporated byreference.

The structure of TAXOL, shown below, offers many groups capable of beingsynthetically functionalized to alter the physical or pharmaceuticalproperties of TAXOL.

For example, a well known semi-synthetic analog of TAXOL, named Taxotere(docetaxel), has also been found to have good anti-tumor activity inanimal models. Taxotere has t-butoxy amide at the 3′ position and ahydroxyl group at the C10 position (U.S. Pat. No. 5,840,929).

Other examples of TAXOL derivatives include those mentioned in U.S. Pat.No. 5,840,929 which are directed to derivatives of TAXOL having theformula:

wherein R¹ is hydroxy, —OC(O)R^(x), or —OC(O)OR^(x); R² is hydrogen,hydroxy, —OC(O)R^(x), or —OC(O)OR^(x); R^(2′) is hydrogen, hydroxy, orfluoro; R^(6′) is hydrogen or hydroxy or R^(2′) and R^(6′) can togetherform an oxirane ring; R³ is hydrogen, C₁₋₆ alkyloxy, hydroxy,—OC(O)R^(x), —OC(O)OR^(x), —OCONR⁷R¹¹; R⁸ is methyl or R⁸ and R²together can form a cyclopropane ring; R⁶ is hydrogen or R⁶ and R² cantogether form a bond; R⁹ is hydroxy or —OC(O)R^(x); R⁷ and R¹¹ areindependently C₁₋₆ alkyl, hydrogen, aryl, or substituted aryl; R⁴ and R⁵are independently C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, or -Z-R¹⁰; Zis a direct bond, C₁₋₆ alkyl, or C₂₋₆ alkenyl; R¹⁰ is aryl, substitutedaryl, C₃₋₆ cycloalkyl, C₂₋₆ alkenyl, C₁₋₆ alkyl, all can be optionallysubstituted with one to six same or different halogen atoms or hydroxy;R^(x) is a radical of the formula:

wherein D is a bond or C₁₋₆ alkyl; and R^(a), R^(b) and R^(c) areindependently hydrogen, amino, C₁₋₆ alkyl or C₁₋₆ alkoxy.

Further examples of R^(x) include methyl, hydroxymethyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, chloromethyl,2,2,2-trichloroethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,ethenyl, 2-propenyl, phenyl, benzyl, bromophenyl, 4-aminophenyl,4-methylaminophenyl, 4-methylphenyl, 4-methoxyphenyl and the like.Examples of R⁴ and R⁵ include 2-propenyl, isobutenyl, 3-furanyl(3-furyl), 3-thienyl, phenyl, naphthyl, 4-hydroxyphenyl,4-methoxyphenyl, 4-fluorophenyl, 4-trifluoromethylphenyl, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, ethenyl, 2-propenyl,2-propynyl, benzyl, phenethyl, phenylethenyl, 3,4-dimethoxyphenyl,2-furanyl (2-furyl), 2-thienyl, 2-(2-furanyl)ethenyl, 2-methylpropyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl,cyclohexylethyl and the like.

TAXOL derivatives can be readily made by following the well establishedpaclitaxel chemistry. For example, C2, C6, C7, C10, and/or C8 positioncan be derivatized by essentially following the published procedure,into a compound in which R³, R⁸, R², R^(2′), R⁹, R^(6′) and R⁶ have themeanings defined earlier. Subsequently, C4-acetyloxy group can beconverted to the methoxy group by a sequence of steps. For example, forconverting C2-benzoyloxy to other groups see, S. H. Chen et al,Bioorganic and Medicinal Chemistry Letters, Vol. 4, No. 3, pp 479-482(1994); for modifying C10-acetyloxy see, J. Kant et al, TetrahedronLetters, Vol. 35, No. 31, pp 5543-5546 (1994) and U.S. Pat. No.5,294,637 issued Mar. 15, 1994; for making C10 and/or C7 unsubstituted(deoxy) derivatives see, European Patent Application 590 267A2 publishedApr. 6, 1994 and PCT application WO 93/06093 published Apr. 1, 1993; formaking 7β,8β-methano, 6,7-α,α-dihydroxy and 6,7-olefinic groups see, R.A. Johnson, Tetrahedron Letters, Vol. 35, No 43, pp 7893-7896 (1994),U.S. Pat. No. 5,254,580, issued Oct. 19, 1993, and European PatentApplication 600 517A1 published Jun. 8, 1994; for making C7/C6 oxiranesee, U.S. Pat. No. 5,395,850 issued Mar. 7, 1995; for makingC7-epi-fluoro see, G. Roth et al, Tetrahedron Letters, Vol 36, pp1609-1612 (1993); for forming C7 esters and carbonates see, U.S. Pat.No. 5,272,171 issued Dec. 21, 1993 and S. H. Chen et al., Tetrahedron,49, No. 14, pp 2805-2828 (1993).

In U.S. 5,773,464, TAXOL derivatives containing epoxides at the C₁₀position are disclosed as antitumor agents. Other C-10 taxane analogshave also appeared in the literature. Taxanes with alkyl substituents atC-10 have been reported in a published PCT patent application WO9533740. The synthesis of C-10 epi hydroxy or acyloxy compounds isdisclosed in PCT application WO 96/03394. Additional C-10 analogs havebeen reported in Tetrahedron Letters 1995, 36(12), 1985-1988; J. Org.Chem. 1994, 59, 4015-4018 and references therein; K. V. Rao et. al.Journal of Medicinal Chemistry 1995, 38 (17), 3411-3414; J. Kant et. al.Tetrahedron Lett. 1994, 35(31), 5543-5546; WO 9533736; WO 93/02067; U.S.Pat. No. 5,248,796; WO 9415929; and WO 94/15599.

Other relevant TAXOL derivatives include the sulfenamide taxanederivatives described in U.S. Pat. No. 5,821,263. These compounds arecharachterized by the C3′ nitrogen bearing one or two sulfursubstiuents. These compounds have been useful in the treatment ofcancers such as ovarian, breast, lung, gastic, colon, head, neck,melanoma, and leukemia.

U.S. Pat. No. 4,814,470 discusses TAXOL derivatives with hydroxyl oracetyl group at the C10 position and hydroxy or t-butylcarbonyl at C2′and C3′ positions.

U.S. Pat. No. 5,438,072 discusses TAXOL derivatives with hydroxyl oracetate groups at the C10 position and a C2′ substitutuent of eithert-butylcarbonyl or benzoylamino.

U.S. Pat. No. 4,960,790 discusses derivatives of TAXOL which have, atthe C2′ and/or C7 position a hydrogen, or the residue of an amino acidselected from the group consisting of alanine, leucine, isoleucine,saline, phenylalanine, proline, lysine, and arginine, or a group of theformula:

wherein n is an integer of 1 to 3 and R² and R³ are each hydrogen on analkyl radical having one to three carbon atoms or wherein R² and R³together with the nitrogen atom to which they are attached form asaturated heterocyclic ring having four to five carbon atoms, with theproviso that at least one of the substituents are not hydrogen.

Other similar water soluble TAXOL derivatives are discussed in U.S. Pat.No. 4,942,184, U.S. Pat. No. 5,433,364, and in U.S. Pat. No. 5,278,324.

Many TAXOL derivatives may also include protecting groups such as, forexample, hydroxy protecting groups. “Hydroxy protecting groups” include,but are not limited to, ethers such as methyl, t-butyl, benzyl,p-methoxybenzyl, p-nitrobenzyl, allyl, trityl, methoxymethyl,methoxyethoxymethyl, ethoxyethyl, tetrahydropyranyl,tetrahydrothiopyranyl, dialkylsilylethers, such as dimethylsilyl ether,and trialkylsilyl ethers such as trimethylsilyl ether, triethylsilylether, and t-butyldimethylsilyl ether; esters such as benzoyl, acetyl,phenylacetyl, formyl, mono-, di-, and trihaloacetyl such aschloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl; andcarbonates such as methyl, ethyl, 2,2,2-trichloroethyl, allyl, benzyl,and p-nitrophenyl. Additional examples of hydroxy protecting groups maybe found in standard reference works such as Greene and Wuts, ProtectiveGroups in Organic Synthesis, 2d Ed., 1991, John Wiley & Sons, andMcOmie; and Protective Groups in Organic Chemistry, 1975, Plenum Press.Methods for introducing and removing protecting groups are also found insuch textbooks.

The markers of the present invention are also shown to be sensitive tocis-Diamminedichloroplatinum (II), otherwise known as cisplatin.Cisplatin is a chemical compound within a family of platinumcoordination complexes which are art-recognized as being a family ofrelated compounds. Cisplatin was the first platinum compound shown tohave anti-malignant properties. The language “platinum compounds” isintended to include cisplatin, compounds which are structurally similarto cisplatin, as well as analogs and derivatives of cisplatin. Thelanguage “platinum compounds” can also include “mimics”. “Mimics” isintended to include compounds which may not be structurally similar tocisplatin but mimic the therapeutic activity of cisplatin orstructurally related compounds in vivo.

The platinum compounds of this invention are those compounds which areuseful for inhibiting tumor growth in subjects (patients). More than1000 platinum-containing compounds have been synthesized and tested fortherapeutic properties. One of these, carboplatin, has been approved fortreatment of ovarian cancer. Both cisplatin and carboplatin are amenableto intravenous delivery. However, compounds of the invention can beformulated for therapeutic delivery by any number of strategies. Theterm platinum compounds also is intended to include pharmaceuticallyacceptable salts and related compounds. Platinum compounds havepreviously been described in U.S. Pat. Nos. 6,001,817, 5,945,122,5,942,389, 5,922,689, 5,902,610, 5,866,617, 5,849,790, 5,824,346,5,616,613, and 5,578,571, all of which are expressly incorporated byreference.

Cisplatin and related compounds are thought to enter cells throughdiffusion, whereupon the molecule likely undergos metabolic processingto yield the active metabolite of the drug, which then reacts withnucleic acids and proteins. Cisplatin has biochemical properties similarto that of bifunctional alkylating agents, producing interstrand,intrastrand, and monofunctional adduct cross-linking with DNA.

C. Sensitivity Assays And Identification Of Therapeutic And DrugScreening Targets

A sample of cancerous cells with unknown sensitivity to a giventherapeutic agent is obtained from a patient. An expression level ismeasured in the sample for a gene corresponding to one of the markersidentified in Tables 1-4. If the gene is expressed, and the marker ofthe invention to which the gene corresponds is a sensitivity marker,then the therapeutic agent will be or is likely to be effective againstthe cancer. Accordingly, if a sensitivity marker is not expressed, thenthe therapeutic agent will not be or will not be likely to be effectiveagainst the cancer. If a resistance marker of the invention isexpressed, then the therapeutic agent will not be or will not be likelyto be effective against the cancer. Accordingly, if the resistancemarker is not expressed, then the therapeutic agent will be or is likelyto be effective against the cancer. Thus, by examining the expression ofone or more of the identified markers in a sample of cancer cells, it ispossible to determine which therapeutic agent(s), or combination ofagents, to use as the appropriate treatment agents.

By examining the expression of one or more of the identified markers ina sample of cancer cells taken from a patient during the course oftherapeutic treatment, it is also possible to determine whether thetherapeutic agent is continuing to work or whether the cancer has becomeresistant (refractory) to the treatment protocol. For example, a cancerpatient receiving a treatment of TAXOL would have cancer cells removedand monitored for the expression of a marker. If the expression level ofa sensitivity marker remains substantially the same, the treatment withTAXOL would continue. However, a significant decrease in sensitivitymarker expression or increased expression of a resistance marker, wouldsuggest that the cancer may have become resistant to TAXOL and anotherchemotherapy protocol should be initiated to treat the patient.

Importantly, these determinations can be made on a patient by patientbasis or on an agent by agent (or combinations of agents). Thus, one candetermine whether or not a particular therapeutic treatment is likely tobenefit a particular patient or group/class of patients, or whether aparticular treatment should be continued.

The identified markers further provide previously unknown orunrecognized targets for the development of anti-cancer agents, such aschemotherapeutic compounds, and can be used as targets in developingsingle agent treatment as well as combinations of agents for thetreatment of cancer.

Summary Of The Data Provided In The Tables

The following terms are used throughout the Tables:

“Accession No” corresponds to the GenBank accession number assigned tothe particular sequence (see, for examplehttp://www.ncbi.nlm.nih.gov/Entrez/nucleotide.html). “GI#” refers to theGenBank GI number assigned to that particular sequence (see, supra).“Database hit” refers to the database in which a homologous sequencematch was found e.g. “GenBank” refers to the GenBank database, “EST”refers to the dbEST database (a division of GenBank), and “NUCPATENT”referes to a GENESEQ database available through Derwent(http://www.Derwent.com). All referenced database sequences areexpressly incorporated by reference.

Other Embodiments

The present invention is not to be limited in scope by the specificembodiments described that are intended as single illustrations ofindividual aspects of the invention and functionally equivalent methodsand components are within the scope of the invention, in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All references cited herein, including journal articles, patents, anddatabases are expressly incorporated by reference. TABLE 1 Marker 1GGGACTTTCTTTTTTTTTTATTATTTTTTCGAACAACCCCCCCCACAACTGGAGAGGGTAAATTTCGCCCCCACCATGCTTACCAGGGGGGAGGGGGAATTATGAAACGCTCCCTTAGAGAAAGAAACCGAAACGCCCACCACACCCGAGGAACGCAGGAGGCGCGCAAATTACCCACTCAAAGAGCCGGGGAGGCAGCGTCTAAAAGGGTTTTCCCCCCAAAGGGGGGGGGGGAAAGGCCCCCCCCAAAACCTTCCCGGGGGGGCTTTTAAAAGGGGAAATTCCAACCAAAGGGGGGCCCTTTAATAGGGAATCCCCCCTTGGAAAAAAACTTGGGAAAAAAAAGGGAAAAGGGTTTTC CTGGGAAAAMarker 2 CGGGCAGGTACAGCTGGGAATGCTCTGTGGGCCTTCATTCTCTGTCGACTGAGTGGGCCAGACGGTGGATGACCCTCCATAATATTTAATTGGTTTTGGGCATTAGATTCCCTATCTTTTGCTTAAGACAATATTTTGGTGCTCATTTATAAAATGTCATTACGGAAACCTATACATACTGATACTATACTTTTGGGGGTGACTCTTGAGAGGGATATCATTAGAGTATGAGAAGTGCGGGTGTGACTGTGTGATTGATGTGTGTGAAAAGAGGGAGGGAGAAATTAATACCTCTAGACGCATAAAATAATGCTTGGCACACACCAAACACCTGGAAGATGGTAGTCTGTACTATGATTCTTAGATGCTGACATTGGGTGATAAATATCCCTCTCCCTTTACACACACACAC Marker 3ACTTTTTTTTTTTTTTTTTTTCCACCCCCAAACGACCGGAAACGACGGTTAAGGGCCACAAAACCTCAAAAGCCCACCATTTTTAGGGTTAAGTCATTCGGGGAAGGAAATTGCCCAACCCCCCTAAGGGGGTCCCAATTACAGGGCCCCCCGCCTGGAACTTAATAAAAACAAAACCTTTTTGGGGGCTCGAAAAACAGACCCCATAGGCCCCTTAACCCGGGGTTGAGGTAAACCCCGAGGCCCTGGTTTTTTTAACAAAAAAAGGCCCGTGGGACATCTGATTTCCCCCCCCGTTTAACCCCGAAAAAGCAGGGTTTTTAACCATTTAAAACTTAAAAAAACGGTGAAAACCGTTGGGCCCCAAAAACCTAAAAAATTTCTTTTACCCGAAAAAAATGGGGGCCCCGGGACCTTGG Marker 4CGTACAATTCATATTGTGTCATTCACTTGCTTAAATACTTTCAACACCTCCTCACTTACCTCAGGATAAATTGTAAACTCCTTACTATGAGCAAAAGGCTTCACATGATTTGGGTCCTCTCCAGCCTTATTTTTGGCACCCTGTTCTCTATCCCTCAGACACATTCACAATCCAGCCACAGTGAACTTCTTCAGTTCCCTAACTATCACATCCAGGTCTTCAAACATGTGTGGAACATTTTTTATTGATTCTTTATTAGGGTAGCTCCTATTTATCCTTTAGGTCTCACCTTGGTCATAACTTCTTCCTAGAAAGCCTTCCCTAACTCCCCATCTGCGATATATTGCCCTTGCTATGACCTTACGTAGTACGAATACTGACCGTGAAAGCGGTGCCTCACGATGCTTCTGACCTTTTGGGTGATAAGCATGAGGTGTCAGAGAAGCTACCACAGGGATAACTGGCTTGAGGCGGACAAGC GCTCATAGCGAMarker 5 ACTAGCTATTGTAATAGAATCTGTTGACTCAATTTTGGTGACATTCCTGGTCACTTTTATTTTTGTTGGCCATGATGGTTAATAACTTGTTTGGAGTACAGATTTGAATCTTGACTTTGGTAGTTATTTACTTTGTGATCTTGGCCAAGGTCACTGTGGCTTAAATTTCCCATCTGTAAAATGAGTATAATGATAATGATGATGACAGTGGTAACTTCCTTATAGATTTTTTAAAAATGAAGGTGGAAGAAATTAATACCTGTAAACACATAAAATAATGCTTGGCACAGAGCAAACACCTGAAAATTGTTAGTTTTTATTATTATTCTTAGATGCTCACATTGTTTGATAAATATCCCTCTCCCTTCACACACACACACACACACACACACACACACACACCTNAACATCTCTAAAGATATCCCTCTCAAGAGTCACCCCCAAGAGTATAGTATCTATATGTATA Marker 6CCTTTCAAGCGGCCGCCCGGGCACGTACTGCGGCGGGTAGGCCTAGGATTGTGGGGGCGATGAATGAACCGAACAGATTCTCGTTCATTTTGGTTCTCAGGGATCGTCATCATTTTTATTTTTATGGGCTCTGCTGAGGGTGGCAGGTGGTCCACCGTGATTACTATTCTACGCCGTGACGCTGTGGCTTAGCGCCCGGCGAATGGTGGTAAACACGGAGTACCATCCGGCTGCCTCACGAAGGCGCATAACACTGAAAAGGGTGACGGACCCTGAGAACCAAAATGAACGAGGATCTGTTCGGTTCATTCATTGGCCCCACAATCCTAGGCCTACCCGCCGCAGT Marker 7TGTACTGCTCGGAGGTTGGGTTCTGCTCCGAGGTCGCCCCAACTCGAAATTTTTAATGCAGGTTTGGTAGTTTAGGACCTGTGGTTTTGTTGGGTACAGAGCATCTTGTTCTGGCCCCTTCATGACACAACGGCTATGCTCTGTTCCACCACCAATTCACAACACCTACGATCCTAGAATTACAAAGACATTTTATATGGATTTTGAACAGAATGGAGAGGCAGAGGAACATCTTTGCATTTATACATAATTATCCATCCCTGTTTCACTGATGAGTACCTTCTTTCCAACTTGACCCGACCACACAAGACCCACTCTGGAGCCCTTTATTCCTTTCCAATGGTCCCATAATGGGAATGGCACACAGTGCCTGCCACATGAGGCTGCTAGGTTCATCCTCCTGTACTCTGGCCTGATGAGCTTCTGCTTATGTTCTAACAAGGAATCTGCCTTACACCATGACCCATACAGGGAAAAGA Marker 8GGGCAACTCAGGATTTTAAGTTCCATAAAGTATTGAACCAACTCTAAAATGTTAACTCCTTAATAACAGATTCTCAGTAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTTTTTTTTTTTTTTAACAACCCCCCCCTAGGGGGGGGCTTTTGGGGAAATAAAAAAAGGGGGGGGGGCCCCCCCCCGTGGGGGGGGGGGCCCCCTTCTCCCCTTGGGGGGGATTTTTTTTITAATTGGGGGGG Marker 9ATGCGGAGGGGTCGATGGACGAGAGAGGGTGAGCATGCATGTGGACAAGAGGCTTAAAACCACTACTGTTTCATTTTCACTCACAATTTTGTGAGCCAGGAATTCAGGCACAGCTCAGCAATTACGCTCTACATAATCTTTTACATTTTTAAAAGTAACTGTTAATAAATTGATACGTCCATTCATGAGAAACTTTTAAAAGGAAAAAAAAAAAAAAAAATTTTTGTCCCTTGGCCCCCCCCCCCTTTAGGGGGAATTTGGGGGATTTTCA Marker 10GGCACTTTTCTTTTTTTTTTTTCTTTTTTTTCCAAAAAAAGAGGCGGGGCCCACGGGGTTGCCCTAAACCCAGGCTGGGGGGGAGACCCCCCCCCCCTTTTTGGCCAAAATTACTTATCATACGCGTTCCCCCAAAAGGGAACTGGGGGGGGGGGACACTCTTATAGGCCTCTCCGGAATGGGGGGGGGTTTCTCAAACTACCCCGTTGCCTACACCACAGGAAGCGTTCTATCATCGTGAAGGCTCATTCATTTCTCTGGCAGCAAAAAAATTAATACCCCCCATGATTTGGGAAGCCTTCGGTGGGAAGCGAAAAGGGCTA Marker 11ACCTATATCAGGGAAGTCCGAAGAAGCCTAACGAGTTGAAGGCTAAAGCTGACAGAGAGTGGAAAAGCCAGGCATATTCAATGGCAGTGCTCTCTCTTGACAGATATTCTACTTAATACAGTTACTCACTTTCTTTAACACCATTCCCTGGCATTAGACTTATGTGTAAAACTGGGATAACACCTCCCCAAAACCAACGTGCTTAGAGGAAGTCTTAATAGCCTTAGGAATATCTGATTTAAGAATGTGACAAACCCACTGGGCACTGTGGCTCACCCCTGTAATCCCAGCACTTTGGGAGGCTAATGCGGGCAGATCACTTGAGGCCAAGAGTTCAAGACCAGCATGGCCAACAGAGTGAAACCTCATCTCTACTAAAAATACAGATATTAGCCACGTTTGGTGGCGTGCACCTGTAGTCCCAGCTACTTGTGAGGCTGAGGCACAAGAATTGCTTGAGCCTGAGAGGCACATGTTGCA GTGAGCCGAMarker 12 TGCCGAAGGAGTCAAAGCTGTGAATTATTTCTGCTGCTAGCTGATGAGAGGCCCTTGTGGAAGGGATGGAACAGGTGCGCACAACCGAAGCGTAGAGTCCCACTTGCAACAAAAGGTTACAATATTGTGATGGGCTCTGGCCGGATCTGTTTGTCCAGGTGGACCATCTATATCATCCTACTACTCTGAGCTGTCATTTAATTGCTCATAACAGTACAGATCATGAGTCTCTGGTTGCAAGTCTAACATATATTCATGCAATGTACGTGTATCTGCATGCATGATTACAGC Marker 13ACTTTCTTTTTTTTTTTTTTTTTTTTTGGGAAAAAGGGGACCCCCTCCCGTGGAAAGGACAGAACTGCCCCCTATTTACCGGGACAACCTTTTTATTTAGAGGAGGAGCCGCCCCCCAAAACATTATTTAACTGTTTGGGGGCCCCCCCCGGCTTTAAGGGGGGAAGATGCCCCAGAACAAGAGGGGAATGCTTTCTTTTTAAACGCCAGGCCCCCCCTTTTTTCCCTTTTAAAAAAAAAAAAAGGGGGGGGGATTCCCGAAACCCCCCAAAAAAAAGGCCCTGGGGCGGAACCCCCTTAGGGGGAAATCCCCCCCCCCGGGGGGGCGTAATAAGGGAACCCCCCCCTGGCCCCAAGTTGGGGAAAAAAAGGGGAAAAGGTTTTCCCGGG Marker 14ACTTTTTTTTTCCTTCTTTTTTCCTTTTTTCCGGGGTTCAAGAATGTCCAATCGAGGCACGTGGGGGGTCCTTTGGAGCCAAATCCCGGATTTCCTTGGTTTTTCCGGGAGGAGCTGTACTCAATTGGGCTAACAACACAGGAGCCAAAAACCTGTGTATTATCTCCGCGAAGGGGATCAAGGGCACGGTTGAACACACTTCCCATTGCTGGTGTGGGACACATGGACATGGCCACAGCTAAGAAAGGCAAACCAGACCTCAGAAAAAAGGAACCTA Marker 15ACGCGGGCAAGGAGCCCTACGAGCACACTTCGTTACCATGTAAATAGGAGAGCTTATCACCTGTTATCTATGACCTGCTGATGAATGGTGACCCCTTATCTGCTCAGTATTCCTGCCTGCCCATTCCCTAACACTACCTACTTAGCTAACTATTACTAGCATCAAATACGCTCATGAAACAGATGCCGCTGGAACTATGCTGCGCTCCATGGTCCTATTCCTGATCACCCTTCGCATGCCTACGCATCCTTTACATAACAGACGAGGTCTACGATCCCTCCCTTACCATCAAATCAATTGGCCACCAATGGNC Marker 16GGAACTTTTTTTTTTTTTTTTTTTTTTTTTAGAGAAAAAAAATGTGCCCGGGCCCCAAGCCTGGAAGCCCAGGGGGGGGGGAGACTGAAGTGGACCCCCCCCCTCCGGGGAAAAAAAAAATACCCCCCTGGACCCCAAGGGGAAGGGGGGTCTCCGCCAAAGACCCAGAACCCCGACAAATTTTTTGGCTTTGGGGGAAAAAGGGGGTCTCCCCGGAGGCACAGGGGGGGGAATAACCCCCCCCCCTTAGGGGGAGGCCCCCCCGAGCTGCCAAAAAGGGGGGGGAATACAGGGTGGGACACAGGGCCCGACCCTGAACCTTTAAAGTGGGGTTTTGGGAAGGGGGGTTTAAAAGGGGGACACCCCCGTTCCCCTCCCGGGGGGGCTTTTAAAAGGGGAAATTCCCACCCCCGGGGGGCCGTATATAGGGGACCCCCCCCTGGACCCAAGGTGGGGAAAAAAAGGGGAAAAGGTTTTCCC Marker 17ACTTGTGTTTTTACTCTTTTTTTTTTATTTTCTTTTTTATGGTAATGTATTTTTTTTTGTAACACAAACTGTTTTTAAAAGAAAAGGCTGTTTGAAAAAAAAAAAAAAAAAAAAAGGGAAAAAAAAAAAAAAAAAGGGCAACCCAAAAAAAAAAACCCCCCGGCCAAAAAAACCCAGGGGGGAAAACCCCCACAAGGGGGGAAAAAAAAAAACCCCCCCCCCCCCTAAAAAAAAAAAAGAAAAAACCCCCGCCCCGGCGGCCACGGGGAAAAAAAAGACCCNCCCGGGGGGGGGG Marker 18GACTTTATTTTCTTCTTTTTTTATTTCCAGTGAGTTTTTATCTATTTTGTTGTTTATTTTTTTTGTTTTGTTGAAGACAAGAAAAACTGGCTTTCAAAAAGAATATGGCCCATCCCCCATGGTGAACAGAGGAGGAACGACCACGACCCCCGGGCGGGGGGGAGCTGCTGAACCACATACCCCCGCTGCAGGGGGGGGGGGAGCCCCCGCCCAAGAGGGCATCGAAAAATTCCCACCCCCCCCGTAATTTTTAATAATACTTACAAACACAGGGGGGGGGGGCAAACTTCAGGAACTCTCCAGGGGGAGAGCCCCCCCCCACCATAAGGAGGGTGTTAAGGACTGAAACCGTAATTGCC Marker 19GGCACTTTTTTTTTTTTTTTTTTCAGGCACCGGGATTTAAAGGGGTTTGGGGGCCACCTACAATGTTAACCAAGGTTCTTTTCCTGGACCCCCTCTTTATAACGAGTTTACTAGGATTACCCCCCTAACCATAAAAATTTCCCACATTTTTGAAACCAGACGGGGTGCCTCCTTTCAGCTTAAATATCACACCCCTCTATGTATCAAAGGGGTGAGAATATTGATTCCCCGAGGCAACTAACCTACCGAACCTGTTTTATAGCAAGGGTGTCCGGGATAGAACCTTAGTTTAACTTTAAAGGTGCCCACAGAACCCTTTAACTCCCCTGGCCCCCCCCGGGGGGCCCTTTAAAAGGGGGAATTTTTAAAATTTTCA Marker 20GGGACTTTTTTTTTTTTTTTTTTTTCTTTGACTATTCTTTCTTTCATATTAACTTTTTCTATGTTTACTTGGTTTCATGCTGGTTTAATGGCCATGTTTATAACTTAATGCTAAAAAGCAGAACCCATTTCGTAGGATGGGAGAGACAGATGATTCCATTACACCGATTGGCCAAGGCCGATGGCATCGAATAAAAGAACTTTTGACTGGAGAGAATCACAGATGGGGAATATTTGTCATAAATAAATAATGAAAACCTAAAAAAAGGGGGGGAACCTAAAAAACCCCCCCCCCTTTTTTTTGGGGGGGCCCCCCCCCCCCCAAAAAAAAAAAAAATTTTTTGGGAAAAACCCCCCGGGGATTCTCCCCCCCGGGGGCCCCCCAAAAAGGGGGGATCCCCCCCCCCCGGGGGGGGGAAAAAAAAAACCCCCCCGGGGGCAAAAAATGGGGGAAAAAAAGGGAAAAAATTTTT Marker 21ACTCGGTGGCTTCCCGCGCGACACTGTCTCTCATGCCAAGACTGACTTGTGGGGGAGGGAGGCGGCTAGCGGGCTATAACCCCTTTTGTGGCGGCGGCTGATTTGAAGCGCTTCCAATGACCGGACCCAGAGAAGAGGAAAACTCTACCGGTGCAGGATCACACGGATCAGGTGACCTTGCTCCTTTTGGACTTTTCTTCATTTGTAGATTCAGTATTGGAGCCATCCTAATAAATGTTCAACGCCCTAGATGTTCTTTTTGACATTGACATTATCAATCAACCTGCTCTAAGAGCTGGCTTTGAAATATAGTCTGATGTGTGCCGCACAACATGCCAG Marker 22ACCAAGAAAAATAAAGAAGAGGCTGCAGAATATGCTAAACTTTTGGCCAAGAGAGTGAAGGAGGCTAAGGAGAAGCGCCAGGAACAAATTGCGAAGAGACGCAGACTTTCCTCTCTGCGAGCTTCTACTTCTAAGTCTGAATCCAGTCAGAAATAAGATTTTTTGAGTAACAAATAAATAAGATCAGACTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAGGTCCTTGGGGGCGGCCCCCCCTTGGGGGGAATTTCCCCCCCCGTGGGGGGGGTTTTTTGGGGGACCCAGCCCGGGCACAAAGGGGGGGAAAAAAAGGGAAAAAGGTTTTT Marker 23GGCACTTTGGATCTATAACTTTTTTGTTCTCTGTACGTCGTATACCTTTTGTAGACCTATCTTGCTTTTTTTCAAAAAGCCCATGAAGAAAACACGGGGGGGGAGCTGAAACATTGTGGGCTATCCCCGCGCCCACAAGAAAACCAAAGAAACAACCATATAATAAATCACCCCCGGGGGAGAAAAAACTGGAGATTTTTTTAAAAAAAAAAGGGGGGGCCAAAAAGGGGGGGGGGAAAAAGGGGGGAAAAAATTTTAAAATTTTGGGGGAGGGTTTTAACCCCCCCCTCCCCCCGGGGGGGGGGGTTAAAAAGGGGGAAACCCCCCCCCCGGGGGGGGGAAAAAAAAAAAACCCCCCCCCCCAAAAAAGGGGGGAAAAAAAAGGGGAAAAAGTTTTC Marker 24GACTTTTTTTTTTTTTTTTTTTTTTTTTATCAAAAAAAAAAGAGCCGTTTTTTTTTTTTTCCGAACAAGGGGGAATTTTGGGAAGCGCCCGGGGGCCCAAGGCCTTTCCCAGAGGGGGAACCCCCGGGGGGGCCCCCATATGGGGGGGCAGAGGCCCCGTCCCCCCAAAGGGGGGATCGCCATGCCCCCCAAAATTGGGGGGGAACCCTGTTTAAGCGGGGAAACCGCCTTTTTCCCGAAGCTCTCGAGTCCAACTTTGTGGGGCCCCGGTGCTTTGCCCCCTGTTACTAAAAAAGGGGGGGGGGGTCCTCCTTAAGGGGGGGGCTTGGGGCACGGGGGGCTTCCAAGATGTCCCTGTTGGTGGGGGGGGGGGGAACAAGCCTATCTCTCTTTTGCCCCCAAAAGGGGAAAAAAAAATTTTTTTTTTTCGGGGACCCCCCCCCC Marker 25ACTTGAACTGTGAGGTCATCGAGAATCCCGACACCTGTCCTCATCTGGAACAAGGTAAAAAGGGGTCGCTATGGAGTTCAAAGGACAGAACTCCTGCCTGGTGACCGGGACAACCTGGCCATTCAGACCCGGGGTGGCCCAGAAAAGCATGAAGTAACTGGCTGGGTGCTGGTATCTCCTCTAAGTAAGGAAGATGCTGGAGAATATGAGTGCCATGCATCCAATTCCCAAGGACAGGCTTCAGCATCAGCAAATAAAAAAAAAAAAAAAAAAGGGGTTTCTTCCCCGGGCCCCCCCTAAAAAGGGCAATTTTCCAAAAACCCCCAAAACGGGGGGGCCCCCCCAACTTTGCTTTTAAGGGGCCCAATCCCCCCTTAAGGGAAGGTTTTAAAAAATAAGGGGCCGGGTTTTT Marker 26AACTTTGTTTTTTTTTTATATTTTTTTATAAAAGAACAACCCCCGGGGAAAAGAAGGGGAAAATAGCGCCCCAAATTTTAAACAGGGAAGGGAAAAACGGTTTAAAGAACCCCTTTTAGAAAAAAAACAAAATACCCCCAGACCCGGGGGAAAAAAAGGGGGGACGGGGAAAAACCAAAAAAGAGCGAGGGGGCAAACGTGAAAATGGGTAACCCCCCCCCGAGGGGGCGCCCAACGGGGCGGGGATTATTAAAAAGAAAAAAACCCCCGAGAAACTTAAAAAGGTACGGGGCGGGG Marker 27ACTTTGTTTTCTCTTTTTTTTTTATTTCTTCTTTTTTTTTTTTTTTTATAAAGGGGGAGTCTTTTTTTTCTTTTTTCCCAAAATGACCGGGAAAGCCCTCCCAGGCCCCCCCAGCTTGAGGAGGGGCCCGGCCCCCAAAACCCGCACCCCTTTCTTGCTTGGGGCCTTGAATGAGAAACCCCTTTATCCCCCCCTTTTTGAAAAAAACTCCCCCCCTTTTTTTTTAAAAAAAAAAAAACCCCCCCTTTTTTTTCAAGGAAAATTTTTTTTTGCAAAACCCCCCCCGGGGGGGCCCCCTCCCCCCCCCCCCCCCTGGGGGGAATTCCCCCCCCCCGGGGGCCCTTATTTAAGGACCCCCCCCCCGCCCCCCCTTTGGGAAAAAAAGGGGAAAAATTTTTTCCCG Marker 28ACACTGGGGGCAAATGCGGATAGGTGCCTGTTCTGGCTTAGAACTACAAATCCCAACAAGCATTGACCCTGATCCTAACTGTGTGGCTAGACTCTGTGGAAGGATAGTCTTGGTCGCCCCCCCCGTTAGGAGATAAAGACACTGGCCTTACCTAGGAACTCGTGTTATAAGGCAGGGAAGTCCTGCCACACCGCCGGGGACAGGACTGCAGGGTGGAATATGCCCATGGGCCGGGGGTGGAACCAGACCGTCTCCTGCAAATTTGATGAATATTTCAGTGGGAGCTGAGACCCTGGGTCTGACCCGAGATCTAATGGGTGTGCTCTGAGCATTGGCGACGAGCATGGAGAGAATAAGTGCGTGCCCAACAGCGATGAGAGATACTACGGCTACACTGGGGC Marker 29ACTTTTAACAAAAAAAGCAGGCCCTTCGAAGTTTAGCGTTCTTTTTTCCTCCCCTGTTGCAAATTATCATGGTTAGGGTTGGGATGGAGAAGAGCACGTGTAATATGCGGATGGCACTGACCACGGTGGGCGGACGGACCTGTCTACTCGAAGGCGACCACCTTTAGATCCTGAGACGGGAAGTGGAGGGTGAATAGGTCACCGCGGCCTTTTTTTTTTGTACGTGAACTTCTTGTTGTTTGCTGGGTAAAGATCCTCGAAGGACTTCTGGTTCTTGGTATCCACATCGCCATCCTCATCATGTTCAGCTGCCCGCTTGCCCGTAGTTGACTCAGATTGCTCATCTTCATCTCCATCCTCTTCCTAACCATCACCTTGTTCTTCCTCCTCCTCTTCCTCCCCACCTTTTTCCTTTTTTTCGCCTACCTGATTGAAAGACTCCTGCTCCCCGCGT Marker 30CTCTCCTTTTTTTTCTTTTTTCTTTTTCTCTTTCTGGTGCTGTTTGCCCTTGTTACTTTTTTCTCCTTGCGCCGAAAAAGGCACTGGCCCGTCTTTATTCCTGCCATACGGAGGCCCCTCTGGAGACCCGCTCTGGGGGGCCCCCCCCCCCCCCAAGGTGAGACTTGGCGGCACCACCGATATCCTGCGGGGGGGAAAGGCCCCCCACACAGCAGGCTTTCAAGAAGCGCCCCTTGATGGGGGAGAAAAAAAACTAGCCTATCCCCCCCCCCCAAGAGGGGGGCAATCTCCCCCGGGGAGCCCCCCCAAGGGGGGGGGGCCCCCCCCCCCCCTTTTGGGGGGGAGGGGGGGGGATCCCCCCCCCCCCCCCCCCCGGGGGGGGTAAAAAGGGGAAAACCCCCCCCCGGGGGGGCCTATAAGGGGACCCCCCCCCCCCCCCCCCCCGGGGAAAA Marker 31AGAGAACTTTTTTTTTTATCATTATATTTCCATTTACCTTATTCTTTTTTTAGGTACATTGTAAGTATACTACAACAATTTGTCTCCCTTCAAGGTCCTCTGAGGCAGCAGGCTTTGGGGCTTCTGCTGTCCTTTGGAGGGTGTCTTCTGGGTAGAGGGATGGGAAGGAAGGGACCCTTACCCCCGGCTCTTCTCCTGACCTGCCAATAAAAATTTATGGGCCAAGGGAAAAAAAAAAAAAAAAAAAAAAGGGGAGAAAAAAAAAAAAAAATAAAAAAAAGGGGGGGGGTTTTTAAAACCCCCACCCCCGACACCTCCTGCCCCCCCCCCCCCGCGCCCCCGCCCCCCCCGCGGGGGGGAGAGAACCCCCCCCCCCCCCTGGGGGGAAAAAAAAAAAAAAATAAAACGTTTGTTTTGTTTTTTTAAAAAAAAA Marker 32ACTTAATGTTTTTAATATTTTTGTTTTAAATGAATTTTTAAAAATATATTTTTTTTTTAGAAACATACCTCAAAAAAAGCGCAGGGGGAAAGCAGTTTTCCAAGGGGGATCCCCGCCCCACAAAAAAAAAAAAAAAAGGACTGCATGCTACTGGCTAGAGCGGGGAAAAAAAAAACTAGAACTTTTTTGTACAAAGAAGGGGGGTCACCTGGGAGACCCACCTTGCTGAGAGAAAAAAGCCCATGTTTGGTCCAAAACCCTGGACCAGAGACATTCTGGCCCACCAACTGTGAAAAAAAGGGGCCCAAAAAAAAAAGGATAGAACAAATTTCCCCCTCCGGAGAAAAAAGGGGAGAGTTAAAAAAAAAAAATTTTTTTTTAAAAAAAAAAAAACGGCTTTTGGGTTAAAAAAAAACAAAACTTTTTTTAAATCCAAAAAAAAAACCCCCGCCCCAAACCCCCTTGGGGGGAATTCCCCCC CCCAGGGMarker 33 ACTTTTTTTTTTTTTTTTTTGGGGGGTTTTGTTTTTTTTTAAAAACCCGTTTAAAACCTCCCCCAAGGGGGGGGGGCAGGGGCCAAAAAAAAGCCGGGGGGGACCTTAAACCCCGGGGCTAAAAAAACCCTTTTCCCTTAAAAATTGGGCCCAAAGGCCCTTCCCCCCCCCCGGGGCCTTAAAATTTATTTTGGGGCCAAAAAAAAAAACCCCAGGCCCGGAAAAACCCGGGGTAAAAAAACAAAAGGGGGCTTTGTTTTCCCTTTTACCCACGGGAACCCTTGAGGGCCTTCGCCGCGGGAAAAAGGAAAAAAAAACCCGGTTCCTTTTTTAAAAAAACCCAAAAAAACCTTTGGGAAAAAACCAAACCCCGGGCCGGACCCCCCCTAGGGGGAAATTCCCCCCCCCGGGGGGGCTTTATTGGGGGACCCCACCCGGGCCCCAAGGTGGGGGAAAAAAGGGGAAAAGGGTTTCCCCGGG AAA Marker34 ACAAGCTTATAGCAAAAGGATAAAGGCAAACACCCAACATGACATGCCGCCTGTCCTATGCGTGCGACCATGGGCAAGTTCACGCCTCACGCGCTATGCGGAGCAAATTATGCTCGCGAGTACGTTATGCATTTACATTTATACACGTGTACTCACCAGGGCTGTGCATTGAACAAAAAGATATTCCGCCAAGCCAGATTCGGGCGCTCCCATCTTGCGCAAGTTGGTCACGTGGACACCCAATTCTTTGATGGCTTTCACCTGCTCATTCAAGTAATGTGTCTCAATGAAGTCACACAAATGGGGGTAATTTTTGTCAGCGGCCAGTTTGTGCAGTTCCAGTAGTGACTGATTCACATTTTTTTCCAAATGTAATGCACACTCCATTGCATTCAGCCCGCTCTCCCAGTCATCACAGTCTGGTTTCTTGATATCCTGAAGGAAGATTCGGACACCTCGTTGGTTCTGCAGCTTCATCAGTTTCTCAGCATGTTCCCTCTCCTCATGAG Marker 35ACTTTTTTTTTTTTTCTTTGTTCTCTTTGGGGGGCTCTGGCTTAAGCCCGTAATTGGGCATTTTGGGAGGGGGAGGCACGCTGATTCCCCCAAAGGAGGTAGGGGGGGACCAAACTGGGGAAGAACGGGGAAGCCCAGTCTCTAGCAAAAATACAACACCCAGCCATGTCCCCCGGCATGCATCTGTACTCCTAGGTACTCCCCACGCCCATGTGGGAGGATAGCTTGAGGCTGAAAGCTGGGGACAGCAGTGATCCAATCTGAGACCACTGCACTCCATTTGGAGAAAAAGAGTGAGACCATGTCTCAAAAAATGAAAAATAGAGGGATGTTATAATACCACCCCCCCGGGTTCCTTGGGGCAAAACCCCCTTGGGGGGGATTTGGGAAAAAAAAAAAAAAATGGGCGCCCCCTTTGGGGTTTTCTTTTAGGGGGGCCCCCCCCCCCCAAAAGGGGGGGGTTAAAAAAAACCGGG Marker 36GCAAAAGGTACAAGGTACACCCCAACATGCATGCACTGTCTTGGTGACCAGGCGAAGTCACCCCACGGATGTGGGGAAATTAGCCCGAGGCTTAGCTACCATTATCACTGATCTACCAGGGTGAGCTCGTCAAAGAGATATTCCGCCAAGCCAGACACTGGCGCTCCCATCTTAGCGCAAGTAGGTCACGTGGTCACCCAATTCTTTGATGGCTCTCACCTGCTCATACAGGGAATGTGAATGATTGAAGTCACACTAATGGGGGTCATTTCTGTCAGTGACCAGCCTGTGCAGCAACAGAAGGGACTGATGCCGCGCGTACCT Marker 37CGGACTTTACCATTAATTTCATTTTTCTTTTTCTCTTTCTGGTGCTGAAAAGAAAGGGAACTCTGGCACCTTGGGCTGAGGAAGGCTCTGGAAAGAACTTCAAGGGTGGAGCCCGCCCTCCTAACAAAAGGGGCCAGTGCCTGGAGATACAACCAAACCTGGGGGGCAGAGGGACTGGCCCCCCCCAGGAAGAAGAGAGCCCCCCTGACACTGGGGGAATCAAATGCCTGGAGGGTGAAGACACCTTAAACCCACCAGGAGGAAGCCTGGGAAGTGCCCAGTGACTTATGGCCAATGTTTGAAGAAAAAGGGGGGGGGGTTATGAGAAAAGGATGGCCAGAGCAAGCGTGACTTGAAGCGGTGCCTCCTCATGTGTGGGAGAGGCTGCGAAACCCCTGCGAAAGCTTGATTCCTGCCATA TG Marker38 TACTACTGAGATCCACTTTTCTGGCCTCTGGCCATTGGCTCTGCCTGTGCACTGATGATCATAATAGTAATTGTAGTGGTCCTGTTCCAACATTACCGGTTAAAGCGATGGGCCCAAAGAGCTCATAAAGAGGTGGCCCTCTAATCAAAAGAAGAGGCCTGGCTCAACCGGGAGAAAAAGGTCTCTGTTTATTTAGAAGACACAGACTAACACTCCTAGGAGGGGAGCTGTGATGATTTCCAAGAACACGAACCCTAGTTTTTGTTGAAGGTAATGGAAACTTTA Marker 39ACTCTTTTTTTTCTTTTTTCTTTTCCGCTTTCTGGTGCTTTTTTTCCTTGTGAACAAAAAAAACATGGGCTGTGGAAAGCTCTGGAAAGACCCTCAAAGCTGGAAAAAGACCTCCTAAGA A Marker 40ACTCCATTAAGTTCATCGTTCTTTTTCCCTTTCTGGTGCTGTTTGCCCTTGTAACTTTTGGCACCTGGGCTGAGGAAGGCACTGGAAAGTCTTGATTTCTGCCATATGGAGGAGTCTCTGGAGACCTGCTCTGTGTGGCCCACGTCCTTTCCACCGTGAGACTTGGCTGCACCACTGATATCCTCCTTGGGGGAAAGGCTTGGCACACAGCAGGCTTTCAAGAAGTGCCAGTTGATCAATGAATAAATAAACGAGCCTATTTCTCTTTGCGAAAAAGGGGGGGAAATTTCCCCGGGAAAACCCCCCAAAGGGGGGGAAGAGCCCCCCTTTTTTTTGGGGGGGGGGGGGGGGGTTTCTCCCCNCTTTTTTCTGGGGGGG Marker 41TCAGGCACCTAGAACTGGGGAAGGCTTTGGAAAGTTCTTCAAAGCTGGAGTTTGTCTACTCAAGAAATGTGCCCAGTGCCTTACATACAGGGAAACCTGAGTGCCGGAGCGACTGGCAGTGGTCAGGGAAGAAGAGATGCCGCCCTGACACTTGTGGCATCACATGCCTGGTATGTGGGGACACCCCCTACCCAGGGAGGAGGAAGGC Marker 42AACTTCGTTCTCTATATCTTGCGGTTTGTTTAGGATTTTTTTTTTTCTTTTTCTTTTTAGGTTTTGAGGAAACACAAAAAGAAACGCCTGGTGCAGAGCCCCAATCCCTACTTCATGGATGAGAAATGCCCAGGATGCTATAAAATCACCACGGGACTTTAGCCATGCACAAACGGTTTTTTTTGTGTGTTGGCTGCTCCACTGTCCTGTGCCACCCTACAAGAAGAAAAGCAAGGGGTTACAGGAAAGATGAACCCTTCAGGGAGGAAGCAGCACTAAGAGCACTCTGAGTCAAGAAGAGTGGGAAACCGTCTCAATAAACACATTTTGGATAAATCCTGCCCCCCCCCCCCCGGGGGGGGGGGGGGAAAAAAAATTTTTTGGGGGGGGGGTTTTTTTTTGGGGGGGGGGCCCCCCCCGGAAAAAAACCCGGTTTTTTTGGGAAACCCCCCCCGGGGGGGGGGCCCCCCCCCGGGGGGGGGGGGAGAGGGGTACCCCCCCTGGGCCCCAATTGGGGAAAAAAAGGGGGGTTT Marker 43ACAAGTCACAAAGCGTCTCCAACTTCCATTCCTCTCTCTCAACATTAATATAAAACTGTTTGATTCCTTCAAGGGTCAATTCTTCCTTTTTCACCAGAATTCGAATTGGATCTCTCATGAATTTTTTGGTCACTTCCAACACATCAGTTGGCATTGTGGCAGAAAGCAACACAACCTGAATACTTGTGTTTAGTTTTTGGAAAATCTCATAGATTTGATCCTTAAAACCACGGCTCAACATTTCATCTGCTTCATCCAAAACAAACATTTTGATCTATTTTGGAGAAAGGTATCTTCTGTTTAACATATCAAACACTCTCCCGGGTGTACGCGGGGGCCAATGTTTGATGCTTAACCCCCCCAATTTCTGTGAGATGGATGGCCAGTGCAAGCGTGACTTGAAGTGTTGCATGGGCATGTGTGGGAAATCCTGCGTTTCCCCTGTGAAAGCTTGATTCCTGCCATATGGAGGAGGCTCTGGAGTCCTGCTCTGTGTGGTCCAGGTCCTTTCCACCCTG Marker 44ACTATCTTTGCAACCTTTTAATAAATCTAGAACTCTTCTAAAATGATAAGTTTATTTAGGCCAAGTGTGGTGGCTCGTGCCTACAATCCCAGCACTTAGACTGTTTTTGGCAGGAGGACCACCCAAACCTAGGAGTTGAAGACCAGCCTGGACAACACAGTGAGACCCCATCTTCAAAAAAAGAAAAGTTTATTTAAAAAATAAAAAATTAGTGAGTGAAAGTTTAAAGAGGATATTTGCACAGCCCCAAAATATCTTCTGCAAAATATTTGTTCATTAGAAAGAGACAAAGTCATAACCTTATAATGGAGAAACCTGGCAGATACTACCTTATCCAAATGATCAGGGTTAACAATGCCAGAAGTCAGACATGTCAGCATCATGTACCTGCCCGGGCG Marker 45GTTCATGTTTGCAATACCTAAAAAGAGAAAATCCACTTTTGCTCACTTTATTTTAAATATCATGTTGAGGTTACTATGCAGAGCTACAACATGCCATCTATAGATGCCAGCCCTGGTCAGTTTTGATTCTTAACCCCATGGACTACTTTGCCTTTTTTCTCTGAAAAAAACTAATTTAAGGGGGCCCGGGTATTTTTTAACTGAATTTAACCGAAATTGACGA Marker 46GGAACTTTTTTTTTTTTTTTTTTTTTTGGGGGAACTTTTAAACCGGGGGGGTAAAGAATTAACCCCTTTTAAAATGGGGGGGGGATTTAAAGCCAAAAAGGGGGAAAAAATATTAACTTCCTTAAAACAGGTTTTTTAGCACACCCAAAAAAGGGGCCCCTTTTTGGGAAAAACTATAAGTTGAAGGGGGGATTCAAGGGGGTCCGGGGAAAAATTGAAAGGCCAAAAAAAAATTTTATTTGAACAACAAATTTT Marker 47AGCAACATCCATACTTCAGAATACAGCTGCCAAGGAGACACCTGTTATGCTGTGGGGACTGGATGGGGCATGGCAGGCGGTTATGGCTAACAACACCTTTTGCTTTGAGCATGGGGGTGGAGGACAGTATCTAATATTTGGGTTCCACAATGCCCACGTGGTCAGGCACGGGCTTATTAAGGCCAATCTTACCAGTTGTGTCCCAGGACAGGGTGATCTTAACCTTGATGCCCAGCACACCTTACTCTTCTTGGATATTAGAACTGGCATCTTGCCGCCGCGCTCCGCTGAAAGGAAAGG ACCCCMarker 48 ACTTTTTTTTTTTTTTTTTTTTTTTAAAACCCGGGGGAGAGAGGGGGTTTTTTTTTGGGGGTGCCCCCCCCCCCCCAAAAAAACCAAAAAGCCCCCCAAAGTCCTCAGGCCCGGGGCGGAGACACCCAGGGGGGTTTTTGTTTTTAAAAAAGGGGGGGTTTAAAAGGGATATGGGAAAAAAGGGGGGAAGAAGTTTTAAACCCTTGGAAAAAATTTTTAAAAGGGTGCCGGGGCCCCCCCAATTTGGCCCCGGGCCCGCCCCCCCCTAGGGGGAAATTCCCCCCCCCTGGGGGGGCGTTTATGGGGGAACCCGCCCTGGACCCAAGTTGGGGAAAAAAAGGGGAAAAGGTTTTCCCCGGGGAAAATTTTCCCCCC Marker 49GCTGGAATCTCGCCCTTAGCGTGGTCGCGGACGAGGTACTTTGATGTGTAGAGCGGAAGAATAGTGTTGAATAAACCCAAGATCGGAATGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTTTTTCCCCCCCCCCGAAAAAAAAAAAAAAAAAATTTTTTTAAAAAACCCCCCCCGGGGGGGGGGGGGAAAAAAAAAAGGGGGGCCCCCCCCCTCTCCCCTAGGGAAAAAAAAAAAAATTTTTGGGGGGGTTTTTAAAAAAGAAAAAAAAAAAAA Marker 50AACTTAACCAAGCTTATTATATTTTGGACTAACCCCTATACCTTTTGCATAATGAATTAACTAGAAATAACTTTGCAAGGAGAGCCAAGCTAATACCCCCGAAACCAGAGAGCTACCTAAGAACAGCTAAAAGAGCACACCCGTCTATGTAGGAAAATATGGGAAGATTATAGGTAGAGGCGACAAACCTACCGAGCCTGGTGATAGCTGGTTGTGCAAGACCCCTAATAAAAAAAACCGGGGTTTTTTTTTTGCCCTTTCCCCTTGGGGGCCCAACCGGGGGGGTTTTTAAAAGGGGGA ATTCCMarker 51 ACCTGCAGACGCTACGGAGCGTAAAACAGCCCTCGGGAACTTTAAAAGGCCAAGACCTCATATGCTTCATATACAAGGGCTCCCCACCGGTGGCCATTGTACAATGGTTGACCAAAGGAAAAGCTGTCACCACAAGAGTGTTACAGACATATCTTCCTCGCCCAAGAGAAGACTCATGTCTTCCGAATGGTTCACTATTCTAACTTCCTGCCGGCAACTGAGGACATTGACGACTGCTCGGTGGAGCACTGTGGCTTGGATGTACCCTCTATGTTAAGCACTGCAAGAACGATCCTACAAGCCATACTCCCAGACACTACATACAACGTAATGCGCGCCCTGGCCCTGACTGTGGGCCTG Marker 52CGTACACGCGGCAAAGCCAACGAGAGTGGACGTATCTTACTTAAGGATATCAAGAAACCAGACTGGGATGACTGGGAGAGCGGCCTGAATGCAATGGAGAGTGCATTACATTTGTAAAAAAATGCTGAGTCAAGGCACTACTGGAACTGTACAAACTGGCCACTGACAAAAATGACCCTGATATGAGTGACTTAATTGAGACACAACACCTGCTTGATGCTGTGAAAGACCTCTGATACTTGCGTGACCACATCTACCAACTGGCTCAAGATGGGAACGGACGAATCTGGCTCGGAGGAATTATCTCTTAGACTAGCACACCCGGGCAGACAGATGATAATGGAAGCTAAGCCTGCCGGC TAA Marker53 ACTTTATTTTTTTTTTTTATTTTTTTTTTTTTTTTATCTCCCCCAAGGGGGGGCGGGGGGGCCCATCAAAAAAAACCCCCCCCCTTCTCAAAAAATTTTTAGGGGAAAAAAATCGGAGGAATACGACAAAAAAAGCTGTGTTCCCCCCAAAAAAAAAAAAAGTTTCCCGCCCATCCCCCCAGAATTAATTACCAAAAAAAGGTTTGAAATAAAACCCCCAAAAACCCTATATCTCCCAAAACACCCGGGGGGAGCCCAAAAAAAAAAAAAAACCTTTGGAAAAAAAAAAAAAAAAAAAAAGGGGGGCTAAAAAAAAAAACCCCTGGGCCCCAACCCCCTTAGGGGGAAATCCCCCCCCCCGGGGGGCTTAAAAAAAGAAACCCCCCCCCAAAACAAAGTTGGGAAAAAAAAGGGAAAAAGGTTTTCCCGGGGAAA

TABLE 2 Marker 1CCTTCTTTGCGTTCTTTCTTTTTGTTTCGCGGGCTCTATGAGACTCGCTCGCCTGAATCTTCACCTAGACCCCTGGCTAGCTCATTTTCTTGCACTGTCACTACAACATTAATCTATGTGTCTTAGATAAAATTTAGATCAAGAGTGCGACGAGCTCTGTTCTTCATTGTGCCGACTAGAACTGTGTTGGCATGTTGACAGAAAAAAGCAGGAATCTTGGGAGAAGAAATCCAAAAAAAATTGTTTGTTTTGTCATTCGGATAAAGAGGAAGAGACTTTGCTGAAATTGTATACAGAACTATATTTGGCTTTTTAACTAGGCCAAGATAGATCTTAGTTGATCACAC Marker 2ACTTTTTTTTTTTCTTTTTTTTTTTTTTCCCCGCGGTTTTCCCCCCGGACCCAAGGGGCGGGAAAACCCCCCCTAAACCCCCAAAGGGGAAGGGGAAAACCTCTTAAAAACCTTGGTGGTTTTGGACCCCGTAATTCCGGAAAGACCCCCCATATGGGCCCTTGGGGTTTAAAAGGGGGGGGGCTCGCCAAAAAAACCCTTGAAAAATTTTAAAAGGGGGCTTCTTAAAAGGGAAAGGGCCCCCTAAAAGTTCCCCGGGCCCTGGGAAACCCGAAAACCTGCCGGGAACCGGGGAACCCCCTTGGATTTCCCTTGGGGAAATTTGAAACCCAAAAATACCCTCCCGGGGGGGCCTTTAAAAGGGGCAATTCCCACACTGGGGGGGGCCGTTAATAGGGGAACCCGGCCTGGGACCAAGGTTGGGGAAAAAAAGGGCAAAAGGGGTTCCCGGG Marker 3ACTTTTTTTTTTTTTTTTTTTTTTTTTAGGGGAACAACATTCTTTAACTTCTTTTGCGAAAACACGGGGTTATTTTTTGCGGGGGGGAGAGTGCCCACAGACTGTCGCGAGCAACCAAGGGGGGGCCCCATTTTACAGGGCATAGGGGTGGGCCCAAAAAAACTAGGTACACTGTCTTATTCTTTGGCATCCGGAAAACTGGCCACACCGCCCCCCAATGAACAGCCCGGGGGGACAACCCAGAGGGGGGGTTTATTAAGGCCCCAAAGGTGCGGGTGTCTGAGTTAAAAAAAA Marker 4GGCACTATGATATTATTTTTTTTTTTTGGGTGACCATTGTTTTTTGTTTCAAAGGGGAAACCAGTTTTTGGCACACCCCCACCCTGCCCAAAAAGACCCACCGAGGACTGCGCATTGTGTCCTGTATAGGGGCATGGTTTTCTGGGGGCGCAAAAAAATCTTTTTCCCCCCCTGGGCAGAAAGGCCACCATCCCCCCACTGGGAGGGGGGGGAGAAAAATAAGAGGGGCCATTTCTACCAAACCCCCGACTTTTTGCTGACCAAAAACAATGCCTGGGGGGAGTAACACCCCTTTTTTTTGACCACCAACCCTCTTTTTGGATAAAAAAAATATGGGGGAAGCACCACCGAAGGGGAAAAACCCCCCAGGGGGGGGGGTGTTTTCCAAA Marker 5ACTTTATTGTTTTTTTTTTTTTTTTTAGGGGAGGGGGAATAACTAGAAACCCCTTTTAAAGGGGGGGCGGTTTTAAGGACCCCCTTGGAGGGGGAAAAAGCTTTCTTAACTAACAGCCAAAGGGAGCTCACCCAGGGTAAAAAAAACAATTCTTTTTGGACCAAAAGGTAAAAGCTACAAGGGTATCTAGCGGGGTTATGGGGGAATTTTCAAGATAGAATAATATTTTATTTTTAAAAAAGCCCATAAAACCGTGTAAAGGGGGTTGGCCCCTTACCCGTAAAACCTCCCACGAACTGTTCTTAAAACACGGGGGGAAATTTTTGAAGAGAGAGAAGAAAACTTAACAGGCATAGGGGG CCTAAAMarker 6 GGGACTTGATTTTTTTTTGCCTGTCTTTTTTCCTGGGGGAAAGAAAATGCTTGATGACCCCGGGGAAGGATCTGGCCTAGGCCGGGTGTAGTGGCTTACACCCGCCCTCCCAGGACTAAGGGAGGCCCCGGGAGGCGCCCCACCTGAGGGGGGGAGTTGGAGACCCCCCCCCCCCCCGAGGAAAAATCTCTTTTTTATAAAAAAACAGGGGATTAGCCCCCTGTGGCGCCGGGTGCCTGAAATCCCAGCCACGGGGGAGGCCCCCCCTGGGGAACCCCAAGAACCTGAAAAGAGGGGGGTGCACCCCACCCCCCCCGCGCCGGGGAACTTTTTCCTGGGTGACAAAAAGATTTTTTGACACCCCGGAAAAAAGGGGGCCCTTTTTTTTTCTTCCATCCCCGGGGGGCCCCCCCTTTCCCCCCGGGGGGGGTGTTTAAAGGGGGAATTCCCCCCCCCCCGGGG Marker 7CACTTGGTGATTCTTTGTTTGTATTAATAGGTTCTGTTTTCTTTCTCTATATTCTCTATGCGCTCTTTTTTTTCGTTGCCTTGGAAAAGGGAGTAAATAAGGAACAAACTGCTCCAGCCCGACGGGGGGGGGCAAATTTGTCAAGATCAAGAAAAAAAAGGCGGGGGGGAAGGTAAAGTTCGATGCAGCAGATACCTTTACACCCTGGACATCACTGACAAAGAGAAGGCTGAGAAACTGAAGCAAACCCTGCCCCCCGGTTTGTTTGTGAAGGAACTGAAAAGAACCAGACACACTGATTGGAACTGGAAAAAAAAAAATACTAATAATCCTATTTGGGGGGGACCCAAAAAAGGGGCCCCTGTTTTTTTTGGGGAAAAAAAATTTTTTTCCACAAAAATATCCCCCAAAAACCCCCCCGGGGGGGCCCGGGGTTTTTGGGGGAAACCCCCCCGGGGGGGGGACCCCCCCACCCCGGGG G Marker 8ACTTTGGCCTCTCTGGGATAGAAGTTCTTCAGCAGGCACACAACAGATGACAGTTGCACGAATTTCAACTGCTCATGAGATGGCGGTCAAGATGATAGACAGTATGGTGCACTCACAGTTCGTTTAATCTCCAGTCGCGTCCCTTGGCCAAAGGTGATATAGGGAACTTGTTACTCTGGTGACAGTACGTAAGTTGCATAATCTTCACGTTG Marker 9ACTTTTTTTTTTTTTTTTTATTTTGGGAAAGGGGTATAACTTTTTCCCCCAAGGGGGAGGGGAATATTGGTTAAATGGACCCCCAGCCCCCGGGGTTAAAACATATTTTTTGGCCAAACCCCCGGGGAAACGGGGATTAAAGGGCCCCCCCCCCAGAGGGTGAAAATTTTGGGTTTTTTAAAAAAAAGGGGGTTTCACCATTGTGGCCAAGGGGGGATTAAACCTCTGCACTAAAGGGAAAAAACCCCCTAAACCCTCAAAAGGGGGGGGATTAAAGGCAAAAACCCATGGGCCCTACCCAAAAATTTTTTTTTGGACCCGGGGTTTTAAAGGGGCAGTGAAAAAATTTTTCCCCTCCCCCTGGGAAAAAAAGG Marker 10ACTTTGTTATTCTTTCTTTTCCTTTTTTCTGGGGAAAAGAATTCTTTTTCCACTTTAACCAACATGGCGGAGACCCCAGACAACAACCCCCCCGTGTTTGGGGGGGACCCCAAAGCCAACAAGCACCAGACCCAACGAGCTGTGACCCTTCTTTTTCCCATAGACGCGAACAAGGGGGACACCCTGCCCCCCCCTGATGGAAACAAGATTGCACACCCCCGACTAAATCCGGGGTACGAGGCTTTAAACCCCCCCAACAAAACCGGGGGGGGGGAAACTGATTTCACTTCCTGGGGCCCACTAGGGGACAGAGAACTCTTGACCAAAAAAAAGGGAAAAACTTTTTTTTTTGGGGGGCCCCTTTTGGGGAAAACCCCGGGCCCCTGGGGCCAACCCCCCTTGGGGGGAAATCCCCCCCCCCGGGGGGCCTTTAATAGGGAACCCCCC Marker 11ACTTTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGACCAAAAACCCATTTAGGGGGGAAAAAAAATGGTTTCCAAATTTCCTAAGAAAAAAAAAAAACCCCCCCAAAAAAAATTCCCCTTTTAAAACAACCCAATTTTTTTTTTCCCCAAAAACTTTTGTTCACTGAAAAAACTTTAAAAAAACCCCAAATTCCCCCAAAAAAAAAAGGGGCAAAAAAGGTTTTTTTTAAACACCCCAAAAAAAAAAAAGGGGGAAATTCTGTACTTTAATAGGAAAATTGTTCCTACCTGGCCCCCCCTGGGACCCAGGGAAAATCCCTTCACAAAAGGGGCCCCCTTCATATGGGAAGAGGAAAAAATCCCCCCCCCCCCGGCCCAGGGGAAAACCCC Marker 12CTAGAGCTTATCTCCTATTCCAAATATATCTTGCGCTGATGAAAAACTGAAAAAAGGCACTTGAAAGCTGTTTCTTTAAGATATGGAATGTGTTTTTTATTCTAGCTGGTAATATATAGCTGCACTGAGCGTGAGCAAATCTTATTCAAGGACATCGCGATGCTGAGAAGTTTAGCCGATAACCTGTCCATCTCTACTTGCAACCGTATTAATCAGAAGAGTACTTTTTGAGTACCTATCAACCACAGGGAGTGAATTCATATTAGATTACCTCAGGTTCAGGGAGGAAAAGCTTGGAAGACGCAGAGAAATCCTGCTCTACTCGCCT Marker 13ACTTAGAGTTTTTTTTATTTTTTTTAGAACTTTATTTTTCTTTTATTTTTTTTTATTTTTTATACCCATCTTGTTTATTTTCAAAATGCCCATGTAGAAAACACAGGCGTGGACCTGTGACATTCTGGACTATTTCTGCGCCCATCAAAAAACAAAAGTAACAACCATATAATAAATCACCTCTGGCGGTGAAAAAACTGGAGGTTTTTTTTTAAAAAAAAAGGGGGGGTTAAAAAGGGGGGGGGCAAAAAGGGGGGAAAAATTTTTTTTATTTTAAAAAAAAATTTTTTAGGGGGGGGGGGGCCCCCAAAAATTTTTTTTTGGGGGCCCCCCCCCCGGGGGGGGAACCCCCCCCCCGGGGGGGGGTATAAAAAAAAAACCCCCCCCCCACAAAAGGGGGGAAAAAAAAAGAAAAAAATATTTTCGGAAAAAA Marker 14ACTTTTTTTTTTTTTTTTTTTTTTTTCCCCCCAAAAAAAAATTTTTTTTGGGGAAAGGCGGGGGGAAAACATTTTCAAAAAAAAAAAAGTGGGAAGGGTTTTTGACCAAAAAACCCCAGGGAAACTTTAGGGTGAACCCCAAATTCCCCCCGGGAAAGGGCCCCCCCCTGGGGGAACCGGGAAGGGCCCTAACCTTTTCAGCCCTGGGGGGAGAATTTTTTAATGGTGAAAACACCCTTGGGGGCCCTATGCCCCGGCCCCAAGAATTTAACTTTTTCCCTGGGAATTTTGCCCCCCCGGGGGGAAAAAACATTGCGGATTTTAACCCCCCGGGGGGGGCCCTTTTTTTTCTTTGAAAAA CCC Marker15 GGCCCTTTTTTCTTTCTTTCTTTCTTTTTCAGAGAAAGGCGGTTTTACTCCCTTCATCCCCCCACTCCCAGGATAGTGACCCCCAGCAGGACAGGGGGGCAGGGCCTACAAACTCAGCAGCACCCCCACGCTGGGCAAAGCCCCCTTCGGCAAACACGGAGTCTACCCCTGCGAAGGGACGCGTCAGGGCCTGAACTCGCCCGTCACAGGGAGCTTCTACCCGTCAAAGTGTTACAGGGAGAAGAGCCCCCACCTGCTGCTCAGTTCCAGCCTGACCGGGTCCCATCCTTTGGCCTCTGTCCCTTTTTCCACAGGGGACCTACCCCTATTGCGGGCCTCCAGCTCATCTTTCACCTCACCCCCCTCTTCCTCCTTGGCTTTAATTCTGCTGATGTTGCCCCAGAATGAATGAATAAAGCGACTCTTTACTTACCCCCCCGGGGGGGCCCT Marker 16GACTTTTGTTCTTTTCTTTTTATTTTTTTTGGGGGGGGAGAGAAACACACCCCCACCCATTTTTTGGGCCCCACATTAAAAAAAGCCAAAAAAGCCTAACCGTGAAAAATAATCTTGGGAAATTACACCACCCCCAAGATGAAAAAAAAAGAGGGGCTAAAATTCACCCCCCAAAAAATATGGAGAAAAAAAATGCCACGAACCCCAAGGGGGGGGGGGGACCCCCGGGTGGTGGGATTCCGCCCGACCAGAGGACAAAAAACTTTTTTGTTTCTTACCCACCCCCTTGGAGAATAACAGCGCAAAGGGGGCCTGAAAATCATCCAAAGCTTTGGAAAAGAAACAAAAAATGTAAAAGAGTGAGGGGCCCAAAAAGCCCCCCTCCACGAGGGGGCACCCCCCCCCATGGGCTG Marker 17ACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAAAATTTTTTTTTTTTTTTTCTGGGGGGGAAAAAACCTTTTTTTTCAAAAACCCCCCCCAAAAAGGGTTTTTTAAAAAAAAACGCCCCCCCCCCAAAAAAAAAAGGCAAGAAAAAAAAAAAGGACCACCAAGAGGCCCGCCCCCCCCTTTTTAAAAACCCGGGGGGGGGGGGGAAAAAAAAGGAAAACCGTTTACAAAGGGGGGGGGAAACCCAAAAAAATCGCCCCCCATGATAACAGGTTTCTGCTTGTAAACCCCCCCAAAAAAAAAATTAAGGGGGGGGCCCCCCCCCCCCCCGGGGGCCCAAAAAAAAACCCCCCCCGCCAGGATTTTGGAGTGCACCCAAAAAAAAAAGGAGGATCTGAAAAAAAAGGAGGGGGGGCCTTTTTTGCTTCCCCCCCCCCCCAAAAAAAAGGGAAACCCTTTGGGGGGGGGG Marker 18GGCACTTTTTTCTTTCTTTCTTTCTGGGACAGAGAGAGGTTTTTTATCGCCCTTCCCTCCCACTTTCTCCCAGTAAAGCCCCCAGGAGCGGGCGGGGGGGACAGCACCTACAGCCTCAGCAGCCCCCTGACGCTGACCCCCCCTGACTACAAAGAGGCACAGAGCCTACGCCTGCGAAGGGGCCCATCATGGACCTGAGCTCGCCCGTGGCAAACATCTTCCCTTAAACATAGTGCTAGAGGGAGAATTTTCCCCACCTGCTCCTCAGGCCCAGGCTGACCCCCTCCCATCCTTTGGCCTTTGACCCTTTTTCCACAGGTTACCTACCCCTATTGCGGTCCTCCAGCTCATATTTCACCT CACCCCCCTCMarker 19 CTGTCGAGGTACTAAGGCCTCCCCAACATCTGTGGCCAGGCTGTCAACAAATAAGTCATACTTGTCAACGATCTCCTCCTTGGTAAGCTCGCCATCCTTGTTTTGGTCTGATTCATAGACCAGGTGCCTGGCTTATGCCTCTGCATGATCATAGTCTGAGGGAAGGATACAGTCTTTGGTCTCTTCCTTGTCCATCTTCCCATCACGGTTCTTATCCCTAAACTCAACAAACTGCTCTCGCTCTGTCTTTACCCATTCTGGCTCATCAGTATTCCCATCGTGGCTGCACTTTGTTTTTTTTTTTTTTTTTTGGGGGCCCCCCACAAAAACGGGGGAAGGGGGGTTTTTTTTTGGGGGGACCCCCCGGGGCCAAAAAAAAACCAAAAAAACCCCAAAATTTTCCCCTCCCCGGGGAAAAAAACCAACGGGGCCTTTTTTTTTTAACCCAGGGGGGTTTTAAA Marker 20TCCTGGTGGGGAAACCCATTTCCCATCCTGTGTCTTGGATTTAAAGAAAACCTGTTGCAGATAATGAGTTGTAAATTCAAGGAGGGTGGCTGTTTTGCTGTTCTTTCTCTGCAGTAAACTCTTATGGGGAGTGTGCCTTGGTTATAAGGCAACGCAAAATGGTAGGGTATATCCATGGATGAATGTTCATCACACCCAGTCTAATTCATACCACGTGGCAGGCTCAGCAAACTGAACCACCACAGGTGTCAGAGATACTTGAGAATGACTGGTACTCGTGCGCCTCGCTTCACTTTTCCTCCGCAACCATGTCTGACAAACCCGATATGGCTGATATCGAGAAATTCTATAAGTCGAAACTGATGAAGACAGAGACGCACGAGAAAAATCCACTGCCTTCCAAAGAAACGATTGAACATGAGAAGCAAGCATGCGAATCGTAATGAGGCGTGCGCCGCCAATATGCACTGT Marker 21CTGGCAATTCGCCCTTAGCGGTGGTCGCGGCCGAGGCACAAAGACCCTCTGTCAAGGGTTCAGGCATTTCCACTCTGCCAATGTAGAATTCTATAGCCACATCCCGGAAAGTCAAGCGTCCCTGAGGAAGAGCCATGCCTGGCTCCTTTCCTTTCCTCTTCTGAGCTGCTTCCTCACGTAACATGAGTCTTTAGGAATCAATCCCAAGCTGGAGTGTGGTGGCAAAAACAGGGCTCACTGCAGCCTCCAACTCCTAGCCTCAAGTGATCCTCCTGTCTCAACCTCCCAAGTAGGATTACAAGCATGCGCCGACGATGCCCAGAATCCAGAACTTTGTCTATCACTCTCCCCAACAACCTAGATGTGAAAACAGAATAAACTTCACCCAGAAAACANANNAAAAAAAAAAAA Marker 22TGTACGCGGTGAGTCAGTCTCCACTCAGGACGACAGCATGGACATGAGGGTGCCCCACTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGACGAGCCGG Marker 23GCGCGGGGAGCGCCGCTGGGCCTGCACGTCTCTGTCGAGCAGCGGACGCCGGTCTCTGTTCCGCAGGATGGGGTTTGTTAAAGTTGTTAAGAATAAGGCCTACTTTAAGAGATACCAAGTGAAATTTAGAAGACGACGAGAGGGTAAAACTGATTATTATGCTCGGAAACGCTTGGTGATACAAGATAAAAATAAATACAACACACCCAAATACAGGATGATAGTTCGTGTGACAAACAGAGATATCATTTGTCAGATTGCTTATGCCCGTATAGAGGGGGATATGATAGTCTGCGCAGCGTATGCACACGAACTGCCAAAATATGGTGTGAAGGTTGGCCTGACAAATTATGCTGCAGCATATTGTACTGGGAGATACAGCCATCCACCTTCAGATGTGTCTACGTGCGCTCTGCCATTCAACTCGGAAACTATAAGTAATTCTCAAGAAAGCCCCCATTTTTATAACCTGGCAAAATCTTGTTAATGTCGTTGCTAAAAAATAAATAAAAGCTAGATACTGGAAACCTAACTGCAATG TG Marker24 ACTTTCTTTTTTTTTTTTTTTTTTTTTTGGCCGCGGCGGCCAGGAAAATCTTTTTGGGGGCTCTTTTGGGACCACGGAAAACGCTTCAGACCCTTGGGGGGGGAGAAAAACCCCCCCCCCGGGGGCCCACTAGATTTTAACCCCCCGGGGGGGGGGGGGGGTTTAAAACCCCAAAAAACTTTTGGGGGGGCCCCCTGTTCTCCACAGGGCCTCCCTTAATAGTCTCCCTGGCAATTGGACTTTTGGGGGAAATCCCCCTTTTAAGGGCCCCGGCCCAGGGGGTTTTTGGCCGGGACCCTTGGGCGGAACCCCCCTAGGGGGGAATTCCCCCCCCCGGGGGGCCTTTTATTGGGGATCCCCCCTTGGCCCCAAATTTGGGAAAAAAAGGGGCAAAGGTTTT Marker 25GGGACTTTTGTTTGTTCTTTTTTTGGTTCTTTGTTTGACAGGGCGTCTTTTGTTTTTTTTCCCAAAAAGGGTCTAATTTTTTTTGTTAGTTCTGACCCCTGGGGGTGGGGCCGTAAACCTTTTAAAAAAGAATGTTGGGCCCGCAAAAAAAAAAGCCCGTTGTGTCTGCCCAAGAACCCTTTTGGGCCGCTGGGGCCCCCCGAGCGCCCGGGTTAAGAGGTTTTTTTTTTGGGACAACCCTGAAAAAATTTTGGGTTTTGGCACCAAGACCCCTA Marker 26ACTTTTTTTTTTTTTTTTTTTCCAACCCCAAACGGCCGGAAACGACGGTTAAGGGCCACAAAAGGTTAAAAGCCCACCATTTTTAGGGTTTAGTCATTCGGGGAAGGAAAGTGCCCAACCCCCCTAAGGGGGTCCGAATTACATGGCCCCCCGCCTGGAACTTAATAAAAACAAAACCTTTTCGGGGGCTCGAATAACCGACCCCATAGGCCCCTTAACCCGGGGTTGAGATCAACCCCGAGGCCCTGGTTGTTTTAACAAAAAAAGGCCAGTGGGACATCTCATTTCCCCCCCCGACTAACCCCGAAAAAGCGGGGTTCTTAACCATTTAAACCTTAAAAAAACGTTGAAAACTGTTGAGACCCAAAAACCTAAAAAAATTCTTTTCCCCGAAAAAAATGGGGGCCCCGGGACCCTGGCCCGAAACCCCCTAAGGGGAAATTCCCACCCCTGGGGGCCGTTAATAGGGGAACCCACCCTGGACCCAAACTTGGGGAAAAAAAGG Marker 27GGCACTTTGTCTTCTTACTCTTTCGCATTTTTTATTGTTCTATTGTGGATTGTTTTTTTTTCTGCGGGAGGACCCCCGGGCGGAACAACGGTTTGAAGCTCGCGACTGGGAAAAAAGACAAGGGGGCCTTGGAGACCACCCCCCTGCTCCCTTTTTATAACCCTGTGGAGGGGCTGGGGAACGCCCTCATCAACAGTGGAACCCGGGAGGACTCCACACGCATTGGGCGCGCCGGGACTGGGAGAGGACAGGCT Marker 28ACTTGTGTTTGCTTAACACAAAGTGACTGTTTGGCTTATAAACACATTGAATGCGCTTTATTGCCCATGGGATATGTGGTGTATATCCTTCCAAAAAATTAAAACGAATATTTCGTCATTATATTTATGGAAGAACTGACTTACACATGGCACACTATGTTGCCATGGGTCTTGCCATCTGGGCCACGCACAAGATCATTTTCTCTTGTGCAGAAAAGTTTTCCATTTTGCAAAAGTCTCCGAAATTCATCGCATGTCTCCTTTTCAGCTTCTCTTTTGACTTTTGCTCTGGGTTCAGCTCTTTCTTTTTCTTTTGCTTCTTGCTGGAAGAAGGCTTCCCCGC Marker 29TCATTAAAAAAAATTCTTAGGTCCTTACCAGCTACGTGTCCTATAGCTCATTCCTACTAGCTTTCTTGACAGATTAGCATTCCCAGTGTCCCGTATTGCCGCTCAAATTGCTCTACTATGCTAACTCCTTGTGTAATAGGCTTACCCTACTTGAAACACTTGAACACTTGATTTCTTTAGCTTTGAGGAGATACCATCTCCAGGGGCGCTTTCTTAGTCTTTGCAGGCACCTCTTTCCTTCAATATCTGATCTTTGTATTGATGAACAAGTTTGGTGGAAACTGCCTTGACCTGTGTCAGCTGGCTCAGCTGATCTCATTTGCTTGCATGGTTTCCCTACCATTTATGTGCGAATGACTGGGAGATTCATATCTGCCTTCTAGATCTTTCCTAATGGATGTGTCTAATTGCTAACAAATGCTATCTGCTGTCTAACGCACTA Marker 30ACTTTTTTTTTTTTTTTTTTTTTTTGTTGCGGAAAAGGACATTTTTGTTAGTACCTTCCGCCCCAACTGGACCATAAAAAACCCCAGGGGGGGGGGGGGGGGAGGGGAAAAAAAGGGCGGGGGGCCCCCCATGGGGGGGCGGCCCTCGGGAAAAAAGGGGAGCGGCCCAAGGGGAGGGGGGGGGAGGGAGAAGAACCCGGAACTGGGGGGGGGCACTTGCCCGATAAACACCTATCCTTGGGAAAATTTTTTTGGGGGGGCAAGCGACCGCCCCGGGGGGGGGTCCTCCAGGGCAAAATTTGGGTTACCCCAAAACTGCTTTTTGCCAATAAAGAGGCTCTTGTGGGAGGAGAAGGAATAACCCTTGGGAACCTTTTGGGGAAACCTTTTGGGGGGAAACCCCGGGGGGGGGGGGGTAACCCCCTTCCAAGGGGCCTGGGGGGGAACCCCCTTAGGGGGAAATTCCCCCCCCCGGG Marker 31CACTTTGTTTTTTTTTTTGTTAGTTTTTTTGGGCAAGAAAGCAAGGTTAGTTTTTTCAGCCCCCGCAACACTTTGAAAATTAAAAACACCGGGGGGGGGGGGGGGGGGGGGAAAAACTGCGGGGGGGGCCCCCAGGGGGGGGGGGCCCCCGGGAAAAAAAGGGTCAACGGCCAAGAGGGGGGGGGGGGGAAGGGCCAACACGGGGACCGGGGGGGGGACCTTACCCCTTAAAAACCTTTGGGGGGGGAACCTTTTTGGAGGGGGGACCTACCCCCCTGGGGGGGGTACAGCGGAGGGGAAACCTTGGGGTTCTCCAAATCCGTGGTTTTAAACATGCTGGGGCTGGAGGAGAATGAAGAAATAAACTTAATTTCCCCGTTTAAAAAACTTTTGGGGGGAAAAACCCGGGGGGGGGGGGTTTACCCCTTTAAAGGGGGTTGGGGGGG Marker 32ACTTTTTTTTTTTTTTTTTTTTTTGGGCGGGGGGGCTTACCAACCCCACTTGGGGGGGCAACCAAGTTTTTCCCAAATTTTTCCTGAAAAAAAAAAAACCCCCCCAAAAAAAACGTTCCCTTAAAAAGACACCAGATTTTTTTTTCCCCAAAAATTTTTCTGTTCGTGGAAACCTTGAAAAAAACCCCAATTCTCCCTTAAAAAAAAAGGGGCAAAACAGGTTTTCTTTGACCCCCCCAAATACTGAAGAGGGGCAAATCTGGTTTCTTTAAAGAGAAAATTTTCCCCCCCGCCGCCCCCGGGCTTTCACTGATAAACTCTTAAAACATTGGTCTCCCCCCCCATGGGGGGTGCTAAAACCCCCCCCCCCCCCTGGAAAGGGGAAACCCCCAAGGGGCCCTTTTTAAATTTAAACCCCCCCCGGGGGGCCCTT Marker 33ACTTTTTTTTTTTTTCTTTTTTTTGGGGGGGGCCTTGGGACCCTTTATCTGGGGTGAGACAAAGGTTTTCCGATTTTTTAAGCTAATGACACAAAAGCCACCCAACCTTATTGCTCCTTTAAAATTCTACCAGCTCTGTTTTTCCCCAGGACCTATTGTTGACTGAGGAAATTTGAAAAGAACCCTAATTTTCCTCTAAAAAAAGATGGGTAAAAATTGTTTTTTTTGATCAGTCCATAAATGAAATTTAGGGGAATT Marker 34ACTTTCTTTTTTATCTTTTTTCTTTTACTCGCCCTGAGGGGGGCACTGCGAACATTAGCCCGGACAGGCACACATTAAATCACCCTAGCCCTCGGGGTACTATCAACAAAACTTTTAAAAAGGGGGCTTTAACCTCTCCACCCTT Marker 35GGCACTTTTTTTTATTTTTTTTTATTTTTTTTTTATTTTAATTTTTCTTTTTTTTTTTTTTATTTTTTTAACCCACCTTTTTTATTATCAAATTTCCCATTTTTTGTCCTCAGCCCTGGACCTGTGACATTTTGGACTATTTCTGTGTTTATTTGAAGACAAAAGAAACAACCATATAATAAATCACCTCTTCCGCTGGTAAAAAAAAAAAATTTTTTTTTTTAAAAAAAAAGGGGGGGTAAAAAAAGGGGGCAAAAAAAGGGGGGAAAAAAATTTTATTTTATAAAAAAAAAATTTTTGGGGGGGGGGGCCCCCCCCCCTTTTTTTCCCCCGGGGGGGGGGGTAAAAAAAAAAAAACCCCCCCCCCCCGGGGG

TABLE 3 Accession Marker # No GI # Database hit Cluster Id Marker 59AF086120 3483465 Genbank Marker 60 X63187 32050 Genbank Hs.2719 Marker61 X07819 35798 Genbank Hs.2256 Marker 62 J00196 188242 Genbank Hs.76807Marker 63 AF200465 6690789 Genbank Marker 64 AL117354 6822199 GenbankMarker 65 AB007862 2662084 Genbank Hs.15896 Marker 66 D83767 1913784Genbank Hs.153678 Marker 67 X06234 34772 Genbank Hs.100000 Marker 68D55654 1255603 Genbank Hs.75375 Marker 69 D83776 1228034 GenbankHs.12413 Marker 70 AC002476 2340101 Genbank Hs.155976 Marker 71 L389321008839 Genbank Hs.12272 Marker 72 X13238 1200056 Genbank Hs.74649Marker 73 AF047442 3335139 Genbank Hs.50785 Marker 74 AF114471 4378758Genbank Hs.251754 Marker 75 AF030455 3169829 Genbank Hs.151602 Marker 76Z48950 761715 Genbank Hs.180877 Marker 77 X04098 28338 Genbank Hs.14376Marker 78 X04503 36490 Genbank Hs.251754 Marker 79 U57029 1405357Genbank Hs.103126 Marker 80 AL035411 6136940 Genbank Hs.23582 Marker 81X04470 28638 Genbank Hs.251754 Marker 82 M28204 576472 Genbank Hs.77961Marker 83 L20941 507251 Genbank Hs.62954 Marker 84 M67480 190364 GenbankHs.250655 Marker 85 AL035209 4160217 Genbank Marker 86 AF203815 6979641Genbank Marker 87 L19739 431318 Genbank Hs.195453 Marker 88 AC0060023983570 Genbank Marker 89 AF110368 6502538 Genbank Marker 90 S61826385936 Genbank Marker 91 AL136543 6807646 Genbank Hs.74335 Marker 92AF202321 6729680 Genbank Marker 93 AC004185 3924650 Genbank Marker 94X15187 37260 Genbank Hs.82689 Marker 95 AA101903 1645427 EST Hs.62954Marker 96 AA112179 1664381 EST Hs.62954 Marker 97 AA483675 2212488 ESTHs.25615 Marker 98 R71431 844948 EST Hs.170980 Marker 99 AA5415952288029 EST Hs.251754 Marker 100 AA165522 1741539 EST Hs.61828 Marker101 AA309592 1961962 EST Hs.184108 Marker 102 AA304969 1957296 ESTHs.50785 Marker 103 AA052990 1543990 EST Hs.180877 Marker 104 AA1329921694561 EST Hs.251754 Marker 105 AA706245 2716163 EST Hs.170311 Marker106 AW024040 5877570 EST Hs.101340 Marker 107 AA350282 2002609 ESTHs.236438 Marker 108 AA460826 2185946 EST Hs.236438 Marker 109 AA0634831557293 EST Hs.62954 Marker 110 AI085373 3423796 EST Hs.83532 Marker 111AA088850 1634371 EST Hs.62954 Marker 112 AA493742 2223583 EST Hs.180532Marker 113 AI525581 4439716 EST Marker 114 AA187815 1774009 EST Hs.62954Marker 115 AA583915 2368524 EST Marker 116 AA303450 1956004 EST Hs.62954Marker 117 AA047033 1525068 EST Hs.8997 Marker 118 AI916667 5636522 ESTMarker 119 AI077331 3411739 EST Hs.76807 Marker 120 AA465517 2191684 ESTHs.73818 Marker 121 AI249257 3845786 EST Marker 122 AA342969 1995205 ESTHs.2186 Marker 123 AA015608 1476656 EST Hs.109773 Marker 124 A094771249532 NUCPATENT

TABLE 4 Marker # Accession # GI # Database hit Cluster Id Marker 1AJ228139 4585698 Genbank Hs.5476 Marker 2 E02628 2170856 Genbank Marker3 AF147334 4761685 Genbank Marker 4 X60489 31099 Genbank Hs.275959Marker 5 AL133216 6983473 Genbank Marker 6 AC002060 7143420 GenbankMarker 7 AB019568 3885371 Genbank Hs.181165 Marker 8 AB018305 3882244Genbank Hs.5378 Marker 9 AF092128 5138905 Genbank Marker 10 AC0076855931443 Genbank Marker 11 U14970 550020 Genbank Hs.76194 Marker 12AF028832 3287488 Genbank Hs.180532 Marker 13 L05092 388031 GenbankHs.179943 Marker 14 AJ010442 3954884 Genbank Hs.156110 Marker 15 S74681807062 Genbank Hs.156110 Marker 16 X57801 33699 Genbank Hs.181125 Marker17 X63432 28335 Genbank Hs.180952 Marker 18 D00017 219909 GenbankHs.217493 Marker 19 AC002480 2340098 Genbank Marker 20 AF103774 6179867Genbank Marker 21 L03162 185397 Genbank Marker 22 D87666 1620016 GenbankHs.180532 Marker 23 M15661 337577 Genbank Hs.118857 Marker 24 D13645286008 Genbank Hs.2471 Marker 25 X96754 1237214 Genbank Hs.156110 Marker26 J00241 185938 Genbank Hs.278553 Marker 27 L01411 185924 GenbankMarker 28 M22918 189019 Genbank Hs.77385 Marker 29 AI401162 4244249 ESTMarker 30 AI972556 5769302 EST Hs.5378 Marker 31 AI337979 4074906 ESTHs.76152 Marker 32 AA449113 2163133 EST Hs.5378 Marker 33 AA0812221623070 EST Marker 34 AA622344 2526220 EST Hs.5378 Marker 35 AI9035676493954 EST Hs.181165 Marker 36 AA083473 1625559 EST Hs.119252 Marker 37AA031525 1501489 EST Hs.74267 Marker 38 AI735753 5057277 EST Hs.74267Marker 39 AA329294 1981697 EST Hs.108124 Marker 40 AA555109 2325648 ESTHs.14376 Marker 41 AW367638 6872288 EST Hs.156110 Marker 42 AW4078466926903 EST Hs.181125 Marker 43 AA402796 2056549 EST Hs.181125 Marker 44AA100764 1647117 EST Hs.217493 Marker 45 AA300732 1953300 EST Hs.156110Marker 46 AA156567 1728218 EST Hs.119252 Marker 47 AA102394 1647372 ESTHs.119252 Marker 48 AA158111 1732896 EST Hs.118857 Marker 49 AA3186621970990 EST Hs.156110 Marker 50 AA377319 2029658 EST Hs.156110 Marker 51AW363100 6867750 EST Hs.2471 Marker 52 AA166716 1745163 EST Hs.36927Marker 53 AL134132 6602319 EST Hs.111515 Marker 54 AA554598 2325137 ESTHs.14376 Marker 55 AA284287 1928569 EST Hs.14376 Marker 56 AI7588695152594 EST Hs.14376 Marker 57 AA832139 2905238 EST Hs.156110 Marker 58Z97273 2245253 NUCPATENT

1. A method for determining whether paclitaxel can be used to reduce thegrowth of a tumor, comprising the steps of: a) obtaining a sample oftumor cells; b) determining whether the tumor cells express one or moremarkers identified in Tables 1, 2, 3 or 4, thereby determining whetherpaclitaxel can be used to reduce the growth of a tumor.
 2. The method ofclaim 1, wherein the level of expression is determined by detecting theamount of mRNA that is encoded by the one or more markers present in thesample.
 3. The method of claim 1, wherein the level of expression isdetermined by detecting the amount of protein that is encoded by saidone or more markers present in the sample.
 4. The method of claim 1,wherein expression of one or more sensitivity markers identified inTables 1 and 3 indicates that paclitaxel can be used.
 5. The method ofclaim 1, wherein expression of one or more resistance markers identifiedin Tables 2 and 4 indicates that paclitaxel cannot be used.
 6. A methodfor determining whether paclitaxel can be used to reduce the growth of atumor, comprising the steps of: a) obtaining a sample of tumor cells; b)exposing the tumor cells to paclitaxel; and c) determining the level ofexpression in the tumor cells of one or more markers identified inTables 1, 2, 3, or 4 in the sample exposed to paclitaxel and in a sampleof tumor cells that is not exposed to paclitaxel, thereby determiningwhether paclitaxel can be used to reduce the growth of a tumor.
 7. Themethod of claim 6, wherein the level of expression is determined bydetecting the amount of mRNA that is encoded by the one or more markerspresent in the sample.
 8. The method of claim 6, wherein the level ofexpression is determined by detecting the amount of protein that isencoded by the one or more markers present in the sample.
 9. The methodof claim 6, wherein increased expression in the presence of paclitaxelof one or more of the sensitivity markers identified in Tables 1 and 3indicates that paclitaxel can be used to reduce the growth of the tumor.10. The method of claim 6, wherein increased expression in the presenceof paclitaxel of one or more of the resistance markers identified inTables 2 and 4 indicates that paclitaxel cannot be used to reduce thegrowth of the tumor.
 11. A method for determining whether treatment withpaclitaxel should be continued in a cancer patient, comprising the stepsof: a) obtaining two or more samples comprising cancer cells from apatient during the course of treatment; and b) determining the level ofexpression in the cancer cells of one or more markers identified inTables 1, 2, 3, or 4 in the two or more samples, thereby determiningwhether treatment with paclitaxel should be continued in a cancerpatient.
 12. The method of claim 11, wherein the level of expression isdetermined by detecting the amount of mRNA that is encoded by the one ormore markers present in the sample.
 13. The method of claim 11, whereinthe level of expression is determined by detecting the amount of proteinthat is encoded by the one or more markers present in the sample. 14.The method of claim 11, wherein treatment with paclitaxel should becontinued if the expression level of one or more of the sensitivitymarkers identified in Tables 1 and 3 does not decrease during the courseof treatment.
 15. The method of claim 11, wherein treatment withpaclitaxel should be discontinued if the expression level of one or moreof the sensitivity markers identified in Tables 1 and 3 decreases duringthe course of treatment indicates that treatment.
 16. The method ofclaim 11, wherein treatment with paclitaxel should be continued if theexpression level of one or more of the resistance markers identified inTables 2 and 4 decreases during the course of treatment.
 17. An isolatednucleic acid molecule comprising a nucleotide sequence of SEQ IDNOs:1-88.
 18. A host cell which contains the nucleic acid molecule ofclaim
 17. 19. An isolated polypeptide which is encoded by a nucleic acidmolecule comprising a nucleotide sequence of SEQ ID NOs:1-88.
 20. Anantibody which selectively binds to a polypeptide of claim
 19. 21. Amethod for detecting the presence of a polypeptide of claim 19 in asample comprising: a) contacting the sample with a compound whichselectively binds to the polypeptide; and b) determining whether thecompound binds to the polypeptide in the sample, thereby detecting thepresence of the polypeptide in the sample.
 22. A method for detectingthe presence of a nucleic acid molecule of claim 17 in a samplecomprising: a) contacting the sample with a nucleic acid probe or primerwhich selectively hybridizes to the nucleic acid molecule; and b)determining whether the nucleic acid probe or primer binds to a nucleicacid molecule in the sample, thereby detecting the presence of thenucleic acid molecule in the sample.